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Histochemical analyses of living mouse liver under different hemodynamic conditions by “in vivo cryotechnique”

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Abstract

Although the morphology and molecular distribution in animal liver tissues have been examined using conventional preparation methods, the findings are always affected by the technical artifacts caused by perfusion-fixation and tissue-resection. Using “in vivo cryotechnique” (IVCT), we have examined living mouse livers with histochemical, immunohistochemical and ultrastructural analyses. In samples prepared by IVCT, widely open sinusoids with many flowing erythrocytes were observed under normal blood circulation, and their collapse or blood congestion was seen in ischemic or heart-arrested mice. In contrast, the sinusoidal cavities were artificially dilated by perfusion-fixation, and collapsed by immersion-fixation and quick-freezing (QF) methods of resected tissues. The immunoreactivity of serum albumin and immunoglobulin G and intensity of periodic acid–Schiff-staining in hepatocytes were well preserved with the QF method and IVCT. Furthermore, following tissue resection, serum proteins were rapidly translocated into hepatocytes as demonstrated by immunoreactions on QF tissues frozen 1 or 5 min after resection. Translocation was not observed in IVCT samples, indicating that IVCT could be useful to examine cell membrane permeability of hepatocytes under different pathological conditions. Both dynamic morphology and immunodistribution of soluble components in living mouse livers, reflecting their physiological and pathological states, can be precisely examined by IVCT with higher time-resolution.

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References

  • Bignold LP (2002) Hypothesis for the influence of fixatives on the chromatin patterns of interphase nuclei, based on shrinkage and retraction of nuclear and perinuclear structures. Br J Biomed Sci 59:105–113

    PubMed  CAS  Google Scholar 

  • Braet F, Wisse E (2002) Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: a review. Comp Hepatol 1:1–17

    Article  PubMed  Google Scholar 

  • Chan FL, Inoue S (1994) Lamina lucida of basement membrane: an artefact. Microsc Res Tech 28:48–59

    Article  PubMed  CAS  Google Scholar 

  • Clemens MG, Zhang JX (1999) Regulation of sinusoidal perfusion: in vivo methodology and control by endothelins. Semin Liver Dis 19:383–396

    PubMed  CAS  Google Scholar 

  • Cogger VC, Fraser R, Le Couteur DG (2003) Liver dysfunction and heart failure. Am J Cardiol 91:1399

    Article  PubMed  Google Scholar 

  • Ekataksin W, Kaneda K (1999) Liver microvascular architecture: an insight into the pathophysiology of portal hypertension. Semin Liver Dis 19:359–382

    PubMed  CAS  Google Scholar 

  • Fahimi HD (1967) Perfusion and immersion fixation of rat liver with glutaraldehyde. Lab Invest 16:736–750

    PubMed  CAS  Google Scholar 

  • Fraser R, Bowler LM, Day WA, Dobbs B, Johnson HD, Lee D (1980) High perfusion pressure damages the sieving ability of sinusoidal endothelium in rat livers. Br J Exp Pathol 61:222–228

    PubMed  CAS  Google Scholar 

  • Fujimoto K (1995) Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes. J Cell Sci 108:3443–3449

    PubMed  CAS  Google Scholar 

  • Furukawa T, Ohno S, Oguchi H, Hora K, Tokunaga S, Furuta S (1991) Morphometric study of glomerular slit diaphragms fixed by rapid-freezing and freeze-substitution. Kidney Int 40:621–624

    PubMed  CAS  Google Scholar 

  • Furuta K, Ohno S, Gibo Y, Kiyosawa K (1992a) Ultrastructural studies of hepatocyte cytoskeleton in experimental cholestasis by quick-freezing and deep-etching method. J Gastroenterol Hepatol 7:22–29

    CAS  Google Scholar 

  • Furuta K, Ohno S, Gibo Y, Kiyosawa K, Furuta S (1992b) Three-dimensional ultrastructure of normal rat hepatocytes by quick-freezing and deep-etching method. J Gastroenterol Hepatol 7:486–490

    CAS  Google Scholar 

  • Geerts A (2001) History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin Liver Dis 21:311–335

    Article  PubMed  CAS  Google Scholar 

  • Greenway CV, Stark RD (1971) Hepatic vascular bed. Physiol Rev 51:23–65

    PubMed  CAS  Google Scholar 

  • Hippe-Sanwald S (1993) Impact of freeze substitution on biological electron microscopy. Microsc Res Tech 24:400–422

    Article  PubMed  CAS  Google Scholar 

  • Kellenberger E, Johansen R, Maeder M, Bohrmann B, Stauffer E, Villiger W (1992) Artefacts and morphological changes during chemical fixation. J Microsc 168:181–201

    PubMed  CAS  Google Scholar 

  • Li Z, Terada N, Ohno N, Ohno S (2005) Immunohistochemical analyses on albumin and immunoglobulin in acute hypertensive mouse kidneys by “in vivo cryotechnique”. Histol Histopathol 20:807–816

    PubMed  CAS  Google Scholar 

  • Liao X, Terada N, Ohno N, Li Z, Fujii Y, Baba T, Ohno S (2006) Immunohistochemical study of serum albumin in normal and cadmium-treated mouse testis organs by “in vivo cryotechnique”. Histol Histopathol 21:35–40

    PubMed  CAS  Google Scholar 

  • McCuskey RS (2000) Morphological mechanisms for regulating blood flow through hepatic sinusoids. Liver 20:3–7

    Article  PubMed  CAS  Google Scholar 

  • McCuskey RS, Bethea NW, Wong J, McCuskey MK, Abril ER, Wang X, Ito Y, DeLeve LD (2005) Ethanol binging exacerbates sinusoidal endothelial and parenchymal injury elicited by acetaminophen. J Hepatol 42:371–377

    Article  PubMed  CAS  Google Scholar 

  • McCuskey RS, Ito Y, McCuskey MK, Ekataksin W, Wake K (1999) Morphologic mechanisms for regulating blood flow through hepatic sinusoids: 1998 update and overview. In: Wisse E, Knook DL, de Zanger R, Fraser R (eds) Proceedings of the ninth international symposium on cells of the hepatic sinusoid. Leiden, Kupffer Cell Foundation, pp 129–134

  • McCuskey RS, Reilly FD (1993) Hepatic microvasculature: dynamic structure and its regulation. Semin Liver Dis 13:1–12

    PubMed  CAS  Google Scholar 

  • Naramoto A, Ohno S, Furuta K, Itoh N, Nakazawa K, Nakano M, Shigematsu H (1991) Ultrastructural studies of hepatocyte cytoskeletons of phalloidin-treated rats by quick-freezing and deep-etching method. Hepatology 13:222–229

    Article  PubMed  CAS  Google Scholar 

  • Ohno N, Terada N, Fujii Y, Baba T, Ohno S (2004a) “In vivo cryotechnique” for paradigm shift to “living morphology” of animal organs. Biomed Rev 15:1–19

    Google Scholar 

  • Ohno N, Terada N, Murata S, Katoh R, Ohno S (2005a) Application of cryotechniques with freeze-substitution for the immunohistochemical demonstration of intranuclear pCREB and chromosome territory. J Histochem Cytochem 53:55–62

    Article  CAS  Google Scholar 

  • Ohno N, Terada N, Murata S, Yamakawa H, Newsham IF, Katoh R, Ohara O, Ohno S (2004b) Immunolocalization of protein 4.1B/DAL-1 during neoplastic transformation of mouse and human intestinal epithelium. Histochem Cell Biol 122:579–586

    Article  CAS  Google Scholar 

  • Ohno N, Terada N, Ohno S (2004c) Advanced application of the in vivo cryotechnique to immunohistochemistry for animal organs. Acta Histochem Cytochem 37:357–364

    Article  Google Scholar 

  • Ohno N, Terada N, Tanaka J, Yokoyama A, Yamakawa H, Fujii Y, Baba T, Ohara O, Ohno S (2005b) Protein 4.1 G localizes in rodent microglia. Histochem Cell Biol 24:1–10

    Google Scholar 

  • Ohno S, Baba T, Terada N, Fujii Y, Ueda H (1996a) Cell biology of kidney glomerulus. Int Rev Cytol 166:181–230

    CAS  Google Scholar 

  • Ohno S, Terada N, Fujii Y, Ueda H, Takayama I (1996b) Dynamic structure of glomerular capillary loop as revealed by an in vivo cryotechnique. Virchows Arch 427:519–527

    Article  CAS  Google Scholar 

  • Okumura S, Takei Y, Kawano S, Nagano K, Masuda E, Goto M, Tsuji S, Michida T, Chen SS, Kashiwagi T, Fusamoto H, Kamada T, Sato N (1994) Vasoactive effect of endothelin-1 on rat liver in vivo. Hepatology 19:155–161

    Article  PubMed  CAS  Google Scholar 

  • Pannen BH, Al-Adili F, Bauer M, Clemens MG, Geiger KK (1998) Role of endothelins and nitric oxide in hepatic reperfusion injury in the rat. Hepatology 27:755–764

    Article  PubMed  CAS  Google Scholar 

  • Quintana AB, Guibert EE, Rodriguez JV (2005) Effect of cold preservation/reperfusion on glycogen content of liver. Concise review. Ann Hepatol 4:25–31

    PubMed  CAS  Google Scholar 

  • Reynoso-Paz S, Coppel RL, Mackay IR, Bass NM, Ansari AA, Gershwin ME (1999) The immunobiology of bile and biliary epithelium. Hepatology 30:351–357

    Article  PubMed  CAS  Google Scholar 

  • Rockey DC (2001) Hepatic blood flow regulation by stellate cells in normal and injured liver. Semin Liver Dis 21:337–349

    Article  PubMed  CAS  Google Scholar 

  • Rockey DC (2003) Vascular mediators in the injured liver. Hepatology 37:4–12

    Article  PubMed  CAS  Google Scholar 

  • Rosser BG, Gores GJ (1995) Liver cell necrosis: cellular mechanisms and clinical implications. Gastroenterology 108:252–275

    Article  PubMed  CAS  Google Scholar 

  • Saxena R, Theise ND, Crawford JM (1999) Microanatomy of the human liver-exploring the hidden interfaces. Hepatology 30:1339–1346

    Article  PubMed  CAS  Google Scholar 

  • Schaart G, Hesselink RP, Keizer HA, van Kranenburg G, Drost MR, Hesselink MK (2004) A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections. Histochem Cell Biol 122:161–169

    Article  PubMed  CAS  Google Scholar 

  • Shiurba R (2001) Freeze-substitution: origins and applications. Int Rev Cytol 206:45–96

    Article  PubMed  CAS  Google Scholar 

  • Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA (1986) Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103:755–766

    Article  PubMed  CAS  Google Scholar 

  • Terada N, Kato Y, Fuji Y, Ueda H, Baba T, Ohno S (1998) Scanning electron microscopic study of flowing erythrocytes in hepatic sinusoids as revealed by ‘in vivo cryotechnique’. J Electron Microsc 47:67–72

    CAS  Google Scholar 

  • Terada N, Ohno N, Li Z, Fujii Y, Baba T, Ohno S (2005) Detection of injected fluorescence-conjugated IgG in living mouse organs using “in vivo cryotechnique” with freeze-substitution. Microsc Res Tech 66:173–178

    Article  PubMed  CAS  Google Scholar 

  • Terada N, Ohno N, Li Z, Fujii Y, Baba T, Ohno S (2006) Application of in vivo cryotechnique to the examination of cells and tissues in living animal organs. Histol Histopathol 21:265–272

    PubMed  CAS  Google Scholar 

  • Terada N, Ohno S (2004) Immunohistochemical application of cryotechniques to native morphology of cells and tissues. Acta Histochem Cytochem 37:339–345

    Article  Google Scholar 

  • Ueno T, Bioulac-Sage P, Balabaud C, Rosenbaum J (2004) Innervation of the sinusoidal wall: regulation of the sinusoidal diameter. Anat Rec A Discov Mol Cell Evol Biol 280:868–873

    Article  PubMed  Google Scholar 

  • van Harreveld A, Biber MP (1962) Conductivity changes in some organs after circulatory arrest. Am J Physiol 203:609–614

    PubMed  Google Scholar 

  • von Schack ML, Fakan S (1994) Retention of glycogen in cryosubstituted mouse liver. Histochemistry 102:451–455

    Article  PubMed  Google Scholar 

  • von Zglinicki T, Rimmler M, Purz HJ (1986) Fast cryofixation technique for X-ray microanalysis. J Microsc 141:79–90

    PubMed  Google Scholar 

  • Wisse E (1970) An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids. J Ultrastruct Res 31:125–150

    Article  PubMed  CAS  Google Scholar 

  • Wisse E, De Zanger RB, Charels K, Van Der Smissen P, McCuskey RS (1985) The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse. Hepatology 5:683–692

    PubMed  CAS  Google Scholar 

  • Yu Y, Leng CG, Kato Y, Ohno S (1997) Ultrastructural study of glomerular capillary loops at different perfusion pressures as revealed by quick-freezing, freeze-substitution and conventional fixation methods. Nephron 76:452–459

    Article  PubMed  CAS  Google Scholar 

  • Zea-Aragon Z, Terada N, Ohno N, Fujii Y, Baba T, Ohno S (2004) Effects of anoxia on serum immunoglobulin and albumin leakage through blood-brain barrier in mouse cerebellum as revealed by cryotechniques. J Neurosci Methods 138:89–95

    Article  PubMed  CAS  Google Scholar 

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Acknowledgement

The authors would like to thank Drs. Takeshi Baba, Yasuhisa Fujii and Zilong Li in Department of Anatomy, University of Yamanashi for many useful suggestions and constructive comments on this work. We also thank Mr. Yutaka Kitahara in Department of Anatomy, University of Yamanashi for his technical help. This study was partly supported by a grant for young investigator from University of Yamanashi to N. Ohno.

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  1. Department of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi, 409-3898, Japan

    Nobuhiko Ohno, Nobuo Terada & Shinichi Ohno

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  1. Nobuhiko Ohno
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  2. Nobuo Terada
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Correspondence to Shinichi Ohno.

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Ohno, N., Terada, N. & Ohno, S. Histochemical analyses of living mouse liver under different hemodynamic conditions by “in vivo cryotechnique”. Histochem Cell Biol 126, 389–398 (2006). https://doi.org/10.1007/s00418-006-0173-6

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