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Three-Dimensional Microvasculature in Rat and Human Hearts Using a Non-injection Ca2+-ATPase Method on Thick and Ultra-Thick Sections

Published online by Cambridge University Press:  22 April 2014

Amaiak Chilingaryan ,
Amayak M. Chilingaryan ,
Mikhail Chilingaryan  and
Gary G. Martin
Amaiak Chilingaryan*
Affiliation:
Department of Biology, Occidental College, 1600 Campus Road, Los Angeles, CA 90041, USA Department of Neurology, LAC+USC Medical Center, 1100 N State St, Los Angeles, CA 90033, USA
Amayak M. Chilingaryan
Affiliation:
Orbeli Institute of Physiology, Yerevan, 0028, Armenia
Mikhail Chilingaryan
Affiliation:
Orbeli Institute of Physiology, Yerevan, 0028, Armenia 221 S. Glendale Avenue, Glendale, CA 91205, USA
Gary G. Martin
Affiliation:
Department of Biology, Occidental College, 1600 Campus Road, Los Angeles, CA 90041, USA
*
*Corresponding author. [email protected]
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Abstract

Currently there are no methods available for staining rat and human myocardial microvasculature on thick sections that would allow for specific staining and differentiation of arterioles, venules, and capillaries. A non-injection technique is described that allows for labeling of the microvascular bed (MVB) in formalin-fixed pieces of the myocardium from humans and the white rat Rattus norvegicus, as well as human full-mount pericardium. Vessel staining is based on the activity of phosphatases (ATPases) and the precipitation of the released phosphate with calcium ions at high pH (pH 10.5–11.5). The resulting precipitate subsequently is converted to black or brown lead sulfide. The specificity of this reaction to vessels of the MVB allows arterioles, venules, capillaries, and pre- and postcapillaries to be clearly visualized in thick (60–100 µm) and ultra-thick (300–500 µm) sections against an unstained background of muscle and connective tissue. In addition, smooth muscle cells of arterioles are also stained allowing for differentiation between arteriolar and venular beds. These observations have not been reported in rat or human myocardium using other methods. This procedure should benefit studies of coronary microcirculation in experimental and pathological conditions, as well as in pharmacological investigations.

Keywords

microcirculationheartATPasecalciumcoronarystaining
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 3 , June 2014 , pp. 895 - 902
Copyright
© Microscopy Society of America 2014 

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References

Anderson, B.G. & Anderson, W.D. (1980). Microvasculature of the canine heart demonstrated by scanning electron microscopy. Am J Anat 158, 217–227.Google Scholar
Andries, L.J., Brutsaert, D.L. & Sys, S.U. (1998). Nonuniformity of endothelial constitutive nitric oxide synthase distribution in cardiac endothelium. Circ Res 82, 195–203.Google Scholar
Badea, C.T., Fubara, B., Hedlund, L.W. & Johnson, G.A. (2005). 4-D micro-CT of the mouse heart. Mol Imaging 4, 110–116.CrossRefGoogle ScholarPubMed
Bell, M.A. & Scarrow, W.G. (1984). Staining for microvascular alkaline phosphatase in thick celloidin sections of nervous tissue: Morphometric and pathological applications. Microvasc Res 27, 189–203.Google Scholar
Bentley, M.D., Qrtiz, M.C. & Ritmam, J.C. (2002). The use of microcomputed tomography to study microvasculature in small rodents. Am J Physiol Regul Integr Comp Physiol 282, R1267–R1279.Google Scholar
Borgers, M., Schaper, J. & Schaper, W. (1971). Localization of specific phosphatase activities in canine coronary blood vessels and heart muscle. J Histochem Cytochem 19, 526–539.Google Scholar
Cappelli, V., Bottinelli, R., Poggesi, C., Moggio, R. & Reggiani, C. (1989). Shortening velocity and myosin and myofibrillar ATPase activity related to myosin isoenzyme composition during postnatal development in rat myocardium. Circ Res 65, 446–457.CrossRefGoogle ScholarPubMed
Chako, K.J. (1977). Nucleoside phosphatase (ATPase) activity in myocardial cells of rat embryos. An electron-microscopic study. Acta Anat (Basel) 97, 379–386.Google Scholar
Chilian, W.M. (1997). Coronary microcirculation in health and disease. Summary of an NHLBI workshop. Circulation 95, 522–528.Google Scholar
Chilingaryan, A.M. (1965). A new method for selective revealing and microscopic study of brain capillaries and vessels. J Exp Clin Med 5, 19–24. in Russian.Google Scholar
Chilingaryan, A.M. (1977). Calcium-ATP method and perspective application of non-injection revealing of organ microvasculatory bed. J Exp Clin Med 5, 19–28. in Russian.Google Scholar
Chilingaryan, A.M. (1986). A new calcium-ATP method for revealing of microvasculatory bed in different organs of cat. Rep Armen Acad Sci 52, 66–71. in Russian.Google Scholar
Chilingaryan, N., Chilingaryan, A. & Chilingaryan, M. (1997). Novel non-injection approaches for revealing and for microscopic investigation of the microcirculatory bed in different organs and tissues. Microsc Microanal Suppl 3, 327–328.Google Scholar
Chilingaryan, A., Chilingaryan, A.M. & Martin, G.G. (2006). The three-dimensional detection of microvasculatory bed in the brain of white rat Rattus norvegicus by a Ca2+-ATPase method. Brain Res 1070, 131–138.Google Scholar
Chilingaryan, A.M. & Martirosyan, J.A. (1973). Study of vascular-capillary networks in mammalian and human skeletal muscles by lead-reactive substances. Armen Biol J 8, 52–57.Google Scholar
Chilingaryan, A.M. & Paravyan, N. (1971). A study of brain capillary proliferation in various periods of postnatal life by means of lead method. Brain Res 28, 550–552.Google Scholar
Djavakhishvili, N. (1997). Significance of sinusoids for myocardial blood circulation. Technol Health Care 5, 171–175.Google Scholar
Fukuhara, T., Kawashima, T., Kubota, I., Mitsunami, K., Motomura, M., Bito, K., Kinoshita, M. & Kawakita, S. (1987). Changes of Ca2+-ATPase and cytochrome oxidase activity of myocardial cell under early and late ischemia: Comparison with ultrastructural changes. Jpn Circ J 51, 403–410.Google Scholar
Gallyas, F. (1970). Silver staining of collagen and reticulin fibres and cerebral capillaries by means of physical development. J Micosc 91, 119–124.Google Scholar
Gallyas, F., Hsu, M. & Buzsaki, G. (1993). Four modified silver methods for thick sections of formaldehyde-fixed mammalian central nervous tissue: “Dark” neurons, perikarya of all neurons, and microglial cells and capillaries. J Neurosci Methods 50, 159–164.Google Scholar
Gao, M., Batra, S., Koyama, T., Yahata, T. & Kuroshima, A. (1997). The capillarity of the subendocardium of left ventricle in rats reared at a low temperature for many generations. Acta Physiol Scand 160, 67–70.Google Scholar
Gao, M., Shirato, H., Miyasaka, K., Kuwabara, M. & Koyama, T. (2000). Induction of growth factors in rat cardiac tissue by X irradiation. Radiat Res 153, 540–547.Google Scholar
Gao, M., Shirato, H., Miyasaka, K. & Koyama, T. (2003). Effect of irradiation on enzymes of the capillary bed in rat ventricles. Adv Exp Med Biol 530, 527–533.Google Scholar
Gossl, M., Malyar, N.M., Beighley, P.E. & Ritman, E.L. (2003). Impact of coronary vasa vasorum functional structure on coronary vessel wall perfusion distribution. Am J Physiol Heart Circ Physiol 285, H2019–H2026.CrossRefGoogle ScholarPubMed
Hossler, F.E. & Douglas, J.E. (2001). Vascular corrosion casting: Review of advantages and limitation in the application of some simple quantitative methods. Microsc Microanal 7, 253–264.Google Scholar
Irino, S., Ono, T. & Shimohara, Y. (1982). Microvascular architecture of the rabbit ventricular walls: A scanning electron microscopic study of corrosion casts. Scan Electron Microsc Pt 4, 1785–1792.Google Scholar
Itoh, J., Kawai, K., Serizawa, A., Yasumura, K., Ogawa, K. & Osamura, R.Y. (2000). A new approach to three dimensional reconstructed imaging of hormone-secreting cells and their microvessel environments in rat pituitary glands by confocal laser scanning microscopy. J Histochem Cytochem 48, 569–578.Google Scholar
Kanaya, H., Akiyama, K., Kitsu, T., Takeyama, Y. & Katagiri, T. (1987). Studies on ultrastructure and cytochemical ATPase activity in human cardiac myocytes from biopsies from patients with various heart diseases. Jpn Circ J 51, 1241–1249.Google Scholar
Kassab, G.S., Lin, D.H. & Fung, Y.C. (1994). Morphometry of pig coronary venous system. Am J Physiol 267, H2100–H2132.Google ScholarPubMed
Kassab, G.S. (2005). Functional hierarchy of coronary circulation: Direct evidence of a structure-function relation. Am J Physiol Heart Circ Physiol 2825, H2559–H2565.Google Scholar
Komaru, T., Kanatsuka, H. & Shirato, K. (2000). Coronary microcirculation: Physiology and pharmacology. Pharmacol Ther 86, 217–261.Google Scholar
Koyama, T., Xie, Z., Gao, M., Suzuki, J. & Batra, S. (1998). Adaptive changes in the capillary network in the left ventricle of rat heart. Jpn J Physiol 48, 229–241.Google Scholar
Lillie, R.D. (1965). Histopathologic Technique and Practical Histochemistry. New York, NY: McGraw-Hill Blakiston Division. pp. 101–103.Google Scholar
Lunkenheimer, P.P. & Merker, H.J. (1976). Distribution of alkaline phosphatase in the microcirculatory pathways of the coronary system. Acta Histochem 57, 14–19.Google Scholar
Malyar, N.M., Gossl, M., Rosol, M., Beighley, P.E. & Ritman, E.L. (2004). Relation of nonperfused myocardial volume and surface area to left ventricular performance in coronary microembolization. Circulation 110, 1946–1952.Google Scholar
Merkus, D., Chilian, W.M. & Stepp, D.W. (1999). Functional characteristics of the coronary microcirculation. Herz 24, 496–508.Google Scholar
Melkonyan, N.N. & Chilingaryan, A.M. (1986). Study of morphofunctional state of organ microvasculatory bed under the influence of pituitrin. Armen Biol J 1, 54–58.Google Scholar
Melkonyan, N.N. & Chilingaryan, A.M. (1990). Effect of chlorpromazine on morphofunctional state of capillary bed in myocardium, pericardium, cerebral cortex and dura mater. Armen Biol J 8, 660–664.Google Scholar
Melkonian, N.N. & Meliksetian, I.B. (2003). Reaction of the brain capillaries to hypoxia in cats. Morfologiia 124, 58–60. in Russian.Google ScholarPubMed
Ono, T., Shimohara, Y., Okada, K. & Irino, S. (1986). Scanning electron microscopic studies on microvascular architecture of human coronary vessels by corrosion casts: Normal and focal necrosis. Scan Electron Microsc Pt 1, 263–270.Google Scholar
Ozawa, K., Takeyama, Y. & Katagiri, T. (1982). Electron microscopic studies on the ATPase activity in myocardial infarction—Changes in the early myocardial infarction. Jpn Circ J 46, 725–733.Google Scholar
Padykula, H.A. & Herman, E. (1955). Factors affecting the activity of adenosine triphosphatase and other phosphatases as measured by histochemical techniques. J Histochem Cytochem 3, 161–169.Google Scholar
Potter, R.F. & Groom, A.C. (1983). Capillary diameter and geometry in cardiac and skeletal muscle studied by means of corrosion casts. Microvasc Res 25, 68–84.Google Scholar
Ratajska, A., Ciszek, B. & Sowinska, A. (2003). Embryonic development of coronary vasculature in rats: Corrosion casting studies. Anat Rec A Discov Mol Cell Evol Biol 270, 109–116.Google Scholar
Samuel, J.L., Perrault, M.A., Bercovici, J., Swynghedauw, B. & Feldmann, G. (1981). Histoenzymological study of myocardial ATPase activity in experimental infarction in the rat. Basic Res Cardiol 76, 602–611.Google Scholar
Schults-Hector, S., Balz, K., Bohm, M., Ikehara, Y. & Rieke, L. (1993). Cellular localization of endothelial alkaline phosphatase reaction product and enzyme protein in the myocardium. J Hitochem Cytochem 41, 1813–1821.Google Scholar
Sellke, F.W. & Dai, H.B. (1993). Responses of porcine epicardial venules to neurohumoral substances. Cardiovasc Res 27, 1326–1332.Google Scholar
Stoker, M.E., Gerdes, A.M. & May, J.F. (1982). Regional differences in capillary density and myocyte size in the normal human heart. Anat Rec 202, 187–191.Google Scholar
Toyota, E., Fujimoto, K., Ogasawara, Y., Kajita, T., Shigeto, F., Matsumoto, T., Goto, M. & Kajiya, F. (2002). Dynamic changes in three-dimensional architecture and vascular volume of transmural coronary microvasculature between diastolic- and systolic-arrested rat hearts. Circulation 105(5), 621–626.Google Scholar
van Beek, J.H. (1997). Is local metabolism the basis of the fractal vascular structure in the heart? Int J Microcirc Clin Exp 17, 337–345.Google Scholar
van Groningen, J.P., Wenink, A.C. & Testers, L.H. (1991). Myocardial capillaries: Increase in number by splitting of existing vessels. Anat Embyol (Berl) 184, 65–70.CrossRefGoogle ScholarPubMed
Wachstein, M. & Meisel, E. (1957). Histochemistry of hepatic phosphatases of a physiologic pH; with special reference to the demonstration of bile canaliculi. Am J Clin Pathol 27, 13–23.Google Scholar
Zinchuk, V.S., Okada, T. & Kobayashi, T. (1999). Ecto-ATPase activity in the rat cardiac muscle: Biochemical characteristics and histocytochemical localization. Cell Tissue Res 298, 499–509.CrossRefGoogle ScholarPubMed
Zinchuk, V.S., Okada, T. & Seguchi, H. (2002). Combined biochemistry and histocytochemistry as a tool to investigate ecto-ATPase in the cardiac muscle. Microsc Res Tech 58, 427–431.CrossRefGoogle ScholarPubMed