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Virchows Archiv B

, Volume 3, Issue 1, pp 229–247 | Cite as

Zum Verhalten der Rattenniere nach Trockeneis-Vereisung

Histologische und enzymhistochemische Untersuchungen
Article

Zusammenfassung

In Anlehnung an kryochirurgisch-experimentelle Arbeiten wurden die Nieren von Albinoratten in der Rindenzone mit Trockeneis vereist und in Abständen von 3 min bis zu 45 Tagen danach histologisch und enzymhistochemisch untersucht.

Durch extra- und intracelluläre Eiskristallbildung mit ihren physiko-chemischen Folgen entsteht eine Coagulationsnekrose aller Tubulusepithelzellen, während interstitielle Bindegewebszellen die Vereisung z.T. überleben. Die unterschiedliche Zellschädigung durch Trockeneis kann mit unterschiedlichem Wassergehalt und/oder andersartiger Permeabilität sowie mit einem gewissen kryophylaktischen Effekt des pericellulären Milieus der interstitiellen Zellen erklärt werden. Nach 1 Std ist die Nekrose morphologisch schon deutlich ausgeprägt, während DPN-Diaphorase, Succinodehydrogenase, Leucinaminopeptidase und alkalische Phosphatase lediglich abgeschwächt sind.

Innerhalb von 24 Std sind alle Tubulusepithelzellen untergegangen, die Basalmembranen sowie das Reticulumfasergerüst bleiben histologisch jedoch erhalten. DPN-Diaphorase und Succinodehydrogenase sind jetzt negativ; Leucinaminopeptidase, unspezifische Esterase, alkalische und saure Phosphatase reagieren dagegen noch positiv. Die positiven Enzymreaktionen in den Tubulusepithelnekrosen werden nicht als Zeichen eines intravitalen Stoffwechsels gewertet.

Im Gegensatz zu anderen Vereisungsmethoden werden nach Trockeneisvereisung kaum Blutungen und keine granulocytären Zellinfiltrate beobachtet. Wahrscheinlich wird durch die gut erhaltenen tubulären Basalmembranen eine chemotaktische Wirkung des nekrotischen Materials auf die Leukocyten verhindert.

Am 4. Tag nach Vereisung ist eine Proliferation der interstitiellen Bindegewebszellen deutlich, die weiter zunimmt und mit Faserbildung und ohne wesentliche Produktion von Mucopolysacchariden einhergeht. Intracanaliculäre Epithelregenerate sind am 6. Versuchstag in den meisten Tubuli zu sehen.

Sowohl Epithelals auch interstitielle Bindegewebszellen weisen histologisch und enzymhistochemisch das Substrat einer intensiven Phagocytose auf. Die Epithelzellen werden dabei offenbar geschädigt und degenerieren etwa vom 12. Versuchstag an zunehmend. Gleichzeitig dringen Bindegewebszellen in die schon am 6. Tag größtenteils verkalkten Tubulusnekrosen ein und bauen diese nur langsam ab.

Am 45. Tag nach Vereisung ist eine sklerosierende und schrumpfende Narbe zu sehen, in der jedoch immer noch reichlich Nekrosen liegen. Die Bildung eines capillarreichen Granulationsgewebes ist im Verlaufe der Narbenbildung nicht erkennbar.

Behaviour of the rat kidney after freezing with dry ice

Histological and enzyme histochemical observations

Summary

Based on methods described in cryo-surgical and cryo-experimental publications, the kidneys of 105 rats were frozen superficially with dry ice (−78.5° C). Within three minutes up to 45 days after freezing the kidneys the rats were sacrificed and their kidneys removed and stained as follows: hematoxylin-eosin, van Gieson, Gomori’s reticular fiber staining, Azan, PAS, alcian blue, methylgreen-pyronin, Sudan III, von Kossa, Berlin blue for iron, acid and alkaline phosphatase, non-specific esterase, succinic dehydrogenase, DPN-diaphorase, and leucine aminopeptidase.

The physico-chemical alterations during dry ice-freezing led to a coagulative necrosis of all tubule cells, whereas some of the interstitial cells resisted the freezing procedure. The different degree of cell damage may be the consequence of a difference in the water content of the cytoplasm and/or of a divergent cell membrane permeability. Moreover, the pericellular fluid of the interstitial cells is believed to have a cryophylactic effect.

One hour after freezing the necrosis was clearly recognizable but the DPN-diaphorase, succinic dehydrogenase, leucine aminopeptidase and alkaline phosphatase exhibited only a reduced reaction.

Within 24 hours after freezing all the tubule cells were necrotic. The basement membranes and the reticular lattice fibers retained their original structures. The reactions for DPN-diaphorase and succinic dehydrogenase were negative. Leucine aminopeptidase, non-specific esterase, acid and alkaline phosphatase were positive in necrotic tubule cells. It is assumed that these positive reactions were not evidence of anin vivo activity.

In contrast to other freezing techniques at lower temperatures after dry ice freezing hemorrhages occurred only in a few cases and there was never an infiltration of the necrosis by leucocytes. It is believed that the well-preserved basement membranes of the tubules prevented the chemotactic effect of the necrotic tubule cells.

Four days after freezing a proliferation of mostly fibroblast-like connective tissue cells was remarkable in the interstices. This proliferation increased during the following days and was accompanied by production of connective tissue fibers whereas mucopolysaccharides were rare.

On the 6th day epithelial cells of most tubules regenerated. Both the epithelial cells and the interstitial connective tissue cells exhibited the substrate of intense phagocytosis. The epithelial cells were altered by the materials they took up and showed increasing degeneration from about the 12th day to the 45th day after freezing. Simultaneously, connective tissue cells invaded the tubules which were for the most part calcified by the 6th day.

On the 45th day after freezing one observed contracted scars rich in fibers. Necrotic and calcified material were still retained.

Formation of a typical granulation tissue with proliferation of capillaries was not observed at any time.

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Literatur

  1. Asahina, E.: Freezing process and injury in isolated animal cells. Fed. Proc.24, Suppl. 15, 183–187 (1965).Google Scholar
  2. Ashwood-Smith, M. J.: Blood and bone marrow preservation. Fed. Proc.24, Suppl. 15, 299–310 (1965).Google Scholar
  3. Bertelli, A.: Proteases and antiproteatic substances in the inflammatory response. In:J. C. Houck andB. K. Forscher, Chemical biology of inflammation, p. 229–240. Oxford: Pergamon Press 1968.Google Scholar
  4. Bickis, I. J., andI. W. D. Henderson: Preservation of dog kidney metabolism by internal freezing and thawing with helium. Cryobiology2, 314 (1966).Google Scholar
  5. Blonde, D. J., E. J. Kresack, andG. W. Kosicki: The effects of ions and freeze-thawing on supernatant and mitochondrial malate dehydrogenase. Canad. J. Biochem.45, 641–650 (1967).CrossRefGoogle Scholar
  6. Breining, H., W. Lutzeyer, S. Lymberopoulos u. S. Langer: Morphologie der Kältenekrose an der Niere. In Vorbereitung.Google Scholar
  7. Buss, H., u.G. Bahre: Zur Wundheilung der Rattenniere nach experimentellem Kälteschaden. Befunde an den Tubuli. Vortrag, Tagung d. Nord- und Westdeutschen Pathologen v. 11. 10‥-13. 10. 1968 in Bielefeld.Google Scholar
  8. —, u.W. Gusek: Untersuchungen über die interstitiellen Zellen der Nierenrinde. Ein Beitrag zur Frage der Matrix mesenchymaler Nierengeschwülste der Ratte. Virchows Arch. Abt. B Zellpath.1, 251–268 (1968).Google Scholar
  9. Chilson, O. P., L. A. Costello, andN. O. Kaplan: Effects of freezing on enzymes. Fed. Proc.24, Suppl. 15, 55–65 (1965).Google Scholar
  10. Cooper, I. S.: Cryogenic surgery of the basal ganglia. J. Amer. med. Ass.181, 600–604 (1962).Google Scholar
  11. Cossel, L.: Die menschliche Leber im Elektronenmikroskop. Untersuchungen an Leberpunktaten. Jena: Fischer 1964.Google Scholar
  12. Cox, R. P., andM. J. Griffin: Alkaline Phosphatase. I. Comparison of the physical and chemical properties of enzyme preparations from mammalian cell cultures, various animal tissues, and Escherichia coli. Arch. Biochem.122, 552–562 (1967).CrossRefGoogle Scholar
  13. Ditscherlein, G., u.R. Natusch: Morphologische Folgen der Nierenblindpunktion am Beispiel der Kaninchenniere. Klin. Wschr.43, 1238–1239 (1965).PubMedCrossRefGoogle Scholar
  14. Eder, M.: Histochemische Fermentnachweise. Ihre Bedeutung in der Pathologie. Verh. dtsch. Ges. Path.42, 374–394 (1958).Google Scholar
  15. Farrant, J., C. A. Walter, andJ. A. Armstrong: Preservation of structure and function of an organized tissue after freezing and thawing. Proc. roy. Soc. B168, 293–310 (1967).Google Scholar
  16. Fishbein, W. N., andJ. L. Griffin: Correlated ultrastructural and enzymatic assay of freeze-thaw damage to mitochondrial isolates. J. Cell Biol.39, 44a (1968).Google Scholar
  17. Fraser, J., andW. Gill: Observations on ultra-frozen tissue. Brit. J. Surg.54, 770–776 (1967).PubMedCrossRefGoogle Scholar
  18. Gage, A. A., G. Fazekas, andE. E. Riley: Freezing injury to large blood vessels in dogs. With comment on the effect of experimental freezing of bile ducts. Surgery61, 748–754 (1967).PubMedGoogle Scholar
  19. Gössner, W.: Untersuchungen über das Verhalten der Phosphatasen und Esterasen während der Autolyse. Virchows Arch. Abt. A path. Anat.327, 304–313 (1955).Google Scholar
  20. Gonder, M. J., W. A. Soanes, andS. Shulman: Cryosurgical treatment of the prostate. Invest. Urol.3, 372 (1966).PubMedGoogle Scholar
  21. Greiff, D., andR. T. Kelly: Cryotolerance of enzymes. I. Freezing of lactic dehydrogenase. Cryobiology2, 335–341 (1966).PubMedCrossRefGoogle Scholar
  22. Gritzka, T. L., andB. F. Trump: Renal tubular lesions caused by mercuric chloride. Electron microscopic observations: Degeneration of the pars recta. Amer. J. Path.52, 1225–1278 (1968).PubMedGoogle Scholar
  23. Gusek, W., H. Buss u.G. L. Laqueur: Histologisch-histochemische Untersuchungen am „Interstitiellen Cycasin-Tumor“ der Rattenniere. Beitr. path. Anat.135, 53–74 (1967).Google Scholar
  24. Halasz, N. A., H. A. Rosenfield, M. J. Orloff, andL. N. Seifert: Whole organ preservation. II. Freezing studies. Surgery61, 417–421 (1967).PubMedGoogle Scholar
  25. Hauschka, T. S., J. T. Mitchell, andD. J. Niederpreum: A reliable frozen tissue bank: viability and stability of 82 neoplastic and normal cell types after prolonged storage at - 78° C. Cancer Res.19, 643 (1959).PubMedGoogle Scholar
  26. Heineke, A.: Die Veränderungen der menschlichen Niere nach Sublimatvergiftung mit besonderer Berücksichtigung der Regeneration des Epithels. Beitr. path. Anat.45, 197–244 (1909).Google Scholar
  27. Holzner, J. H., u.E. Golob: Fermenthistochemische Untersuchungen bei der experimentellen Hydronephrose. Wien. klin. Wschr.80, 167–173 (1968).PubMedGoogle Scholar
  28. Huggins, C. E.: Preservation of organized tissues by freezing. Fed. Proc.24, Suppl. 15, 190–195 (1965).Google Scholar
  29. Jacob, S. W., T. H. Moghul, S. C. Collins, andJ. E. Dunphy: Experimental studies in cryobiology. Amer. J. Surg.102, 254–263 (1961).PubMedCrossRefGoogle Scholar
  30. Janoff, A., andS. Schaefer: Mediators of acute inflammation in leucocyte lysosomes. Nature (Lond.)213, 144–147 (1967).CrossRefGoogle Scholar
  31. Könn, G., u.S. Meiser: Zur morphologischen Pathologie traumatischer Nierenverletzungen und ihrer Spätfolgen. Beitr. path. Anat.137, 350–372 (1968).Google Scholar
  32. Kranz, D., G. Ditscherlein u.J. Kunz: Autoradiographische Untersuchungen zur Wundheilung nach Nierenpunktion der Ratte. Beitr. path. Anat.137, 51–64 (1968).Google Scholar
  33. Leeson, T. S.: An electron microscope study of the postnatal development of the hamster kidney. With particular reference to the intertubular tissue. Lab. Invest.10, 466–480 (1961).PubMedGoogle Scholar
  34. Lovelock, J. E.: Biophysical aspects of the freezing and thawing of living cells. Proc. roy. Soc. Med.47, 60–62 (1954).PubMedGoogle Scholar
  35. — Denaturation of lipid-protein complexes as a cause of damage by freezing. Proc. roy. Soc. Med.147 B, 427–433 (1957).Google Scholar
  36. Lutzeyer, W., S. Lymberopoulos, H. Breining u.S. Langer: Experimentelle Kryochirurgie der Niere. Vortrag, 85. Tagung d. deutschen Gesellschaft f. Chirurgie am 18. 4. 1968 in München.Google Scholar
  37. Lymberopoulos, S.: Die Kryochirurgie der Niere. I. Das „Kryoskalpell“ zur Durchführung kryochirurgischer Parenchymeingriffe (Vorläufige Mitteilung). Urologe7, 224–225 (1968).PubMedGoogle Scholar
  38. Mandeville, A. F., andB. F. McCabe: Some observations on the cryobiology of blood vessels. Laryngoscope (St. Louis)77, 1328–1350 (1967).Google Scholar
  39. Mazur, P.: Causes of injury in frozen and thawed cells. Fed. Proc.24, Suppl. 15, 175–182 (1965).Google Scholar
  40. Melnick, P. J.: Effect of freezing rates on the histochemical identification of enzyme activity. Fed. Proc.24, Suppl. 15, 259–267 (1965).Google Scholar
  41. Meryman, H. T.: Mechanics of freezing in living cells and tissues. Science124, 515–521 (1956).PubMedCrossRefGoogle Scholar
  42. — The mechanisms of freezing in biological systems. In:A. S. Parkes andA. U. Smith, Eecent research in freezing and drying, p. 23–39. Oxford: Blackwell Scientific Publications 1960.Google Scholar
  43. — Modified model for the mechanism of freezing injury in erythrocytes. Nature (Lond.)218, 333–340 (1968).CrossRefGoogle Scholar
  44. Moor, H.: Die Gefrierfixation lebender Zellen und ihre Anwendung in der Elektronenmikroskopie. Z. Zellforsch.62, 546–580 (1964).PubMedCrossRefGoogle Scholar
  45. —, andK. Muehlenthaler: Fine structure in frozen-etched yeast cells. J. Cell Biol.17, 609–628 (1963).CrossRefPubMedGoogle Scholar
  46. Mundth, E. D., A. J. DeFalco, andY. G. Jacobson: Functional survival of kidneys subjected to extracorporal freezing and reimplantation. Cryobiology2, 62–67 (1965).PubMedCrossRefGoogle Scholar
  47. Neumann, K.: Anwendung der Gefriertrocknung für histochemische Untersuchungen. In:W. Graumann undK. Neumann (Hrsg.), Handbuch der Histochemie, Bd. I/l, S. 1–77. Stuttgart: Fischer 1958.Google Scholar
  48. Ortved, W. E., F. M. O’Kelly, I. A. D. Todd, J. B. Maxwell, andM. R. Sutton: Cryosurgical prostatectomy. A report of 100 cases. Brit. J. Urol.39, 577–583 (1967).PubMedGoogle Scholar
  49. Pascoe, J. E.: The survival of the rat’s superior cervical ganglion after cooling to - 76° C. Proc. roy. Soc. B147, 510–519 (1957).Google Scholar
  50. Pearse, A. G. E.: Histochemistry. Theoretical and applied, II. ed. Boston: Little, Brown & Co. 1961.Google Scholar
  51. Polge, C., A. U. Smith, andA. S. Parkes: Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature (Lond.)193, 548–550 (1962).CrossRefGoogle Scholar
  52. Rickles, N. H., J. S. Bennett, andM. A. Hillier: The individual effects of liquid nitrogen and freeze-dry storage on several dehydrogenase reactions in tissue sections. Cryobiology2, 219–222 (1966).PubMedCrossRefGoogle Scholar
  53. Rosen, S., C. L. Pirani, andR. C. Muehrcke: Renal interstitial foam cells. A light and electron microscopic study. Amer. J. clin. Path.45, 32–41 (1966).Google Scholar
  54. Schimmel, H., G. Wajcner, C. Chatelain, andM. Legrain: Freezing of whole rat and dog kidney by perfusion of liquid nitrogen through the renal artery. Cryobiology1, 171–175 (1964).PubMedCrossRefGoogle Scholar
  55. Schoenmackers, J.: Medizinische Fakultät an einer Technischen Hochschule. Erste gemeinsame Schritte. Alma mater aquensis6, 43–52 (1968).Google Scholar
  56. Smith, A. U.: Biological effects of freezing and supercooling. Baltimore: Williams & Wilkins Co. 1961.Google Scholar
  57. — Problems in freezing organs and their component cells and tissues. Fed. Proc.24, Suppl. 15, 196–203 (1965).Google Scholar
  58. Stephenson, J. L.: Ice crystal growth during the rapid freezing of tissues. J. biophys. biochem. Cytol.2, Suppl., 45–52 (1956).PubMedCrossRefGoogle Scholar
  59. Taylor, A. C.: The effect of rate of cooling on survival of frozen tissues. Proc. roy. Soc. B147, 466–475 (1957).CrossRefGoogle Scholar
  60. Trump, B. F., P. J. Goldblatt, C. C. Griffin, V. S. Waravdekar, andR. E. Stowell: Effects of freezing and thawing on the ultrastructure of mouse hepatic parenchymal cells. Lab. Invest.13, 967–1002 (1964).PubMedGoogle Scholar
  61. Trumpf, B. F., D. E. Young, E. A. Arnold, andR. E. Stowell: Effects of freezing and thawing on the structure, chemical constitution, and function of cytoplasmic structures. Fed. Proc.24, Suppl. 15, 144–168 (1965).Google Scholar
  62. Wachstein, M., andE. Meisel: Influence of experimental renal damage on histochemieally demonstrable succinic dehydrogenase activity in the rat. Amer. J. Path.30, 147–165 (1954).PubMedGoogle Scholar
  63. —— A comparative study of enzymatic staining reactions in the rat kidney with necrobiosis induced by ischemia and nephrotoxic agents (mercuhydrin and DL-serine). J. Histochem. Cytochem.5, 204–220 (1957).PubMedGoogle Scholar
  64. Wangensteen, O. H., E. T. Peter, D. M. Nicoloff, A. I. Walder, H. Sosin, andE. F. Bernstein: Achieving “physiological” gastrectomy: by gastric freezing. J. Amer. med. Ass.180, 439–444 (1962).Google Scholar
  65. Waravdekar, V. S., P. J. Goldblatt, B. F. Trump, C. Griffin, andR. E. Stowell: Enzyme activities in rapidly frozen and thawed mouse liver. Fed. Proc.22, 313 (1963).Google Scholar
  66. Ward, P. A. Chemotaxis of polymorphonuclear leukocytes. In:J. C. Houck andB. K. Forscher, Chemical biology of inflammation, p. 99–105. Oxford: Pergamon Press 1968.Google Scholar
  67. Young, D. E., C. C. Griffin, E. A. Arnold, andR. E. Stowell: Quenching mouse liver in isopentane and propane at - 155° C. Some morphologic and chemical effects. Arch. Path.84, 499–508 (1967).PubMedGoogle Scholar
  68. Zimmermann, H.: Experimentelle histologische, histochemische und funktionelle Untersuchungen zur Frage der Nierenschädigung nach temporärer Ischämie. Beitr. path. Anat.117, 65–84 (1957).Google Scholar
  69. Zollinger, H. U.: Niere und ableitende Harnwege. In:W. Doerr undE. Uehlinger, Spezielle Pathologische Anatomie, Bd. 3. Berlin-Heidelberg-New York: Springer 1966.Google Scholar
  70. Zuckermann, A. J., H. E. M. Kay, andA. B. Hockley: Recovery of human foetal liver cells after storage in liquid nitrogen. J. clin. Path.21, 109–110 (1968).CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1969

Authors and Affiliations

  • H. Buss
    • 1
  1. 1.Abteilung Pathologie der Rheinisch-Westfälischen Technischen Hochschule AachenDeutschland

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