Acta Neuropathologica

, Volume 45, Issue 1, pp 47–51 | Cite as

A specific immunofluorescence technique for the demonstration of vasogenic brain edema in paraffin embedded material

  • F. Wilmes
  • K. -A. Hossmann
Original Works


Serum proteins as constituents of vasogenic brain edema were visualized both macroscopically and microscopically applying a double layer immunofluorescence technique to paraffin embedded material derived from three experimental series: peritumorous edema following xeno-transplantation of glioma cells, edema after cerebral embolization with micropheres, and edema after unilateral MCA occlusion. Exclusively cats were used as experimental animals.

The staining procedures resulted in selective green fluorescence of vessel contents as well as edema protein, which was demonstrable even macroscopically at times, where edema formation reaches a maximum in each experimental series. Microscopically, serum protein could be traced up to the end of observation time ranging from 1 to 4 weeks, where the specific fluorescence was related to cellular structures.

As compared to other techniques employed in brain edema localization, immunostaining mainly offers the following advantages: avoidance of in vivo tracing, better structural resolution in paraffin than in freeze sections, high specificity and sensitivity in antigen localization.

Key words

Immunofluorescence Edema Paraffin sections Serum proteins 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Allen, T. H., orahovats, P. D.: Combination of toluidine dyes with plasma albumin. Am. J. Physiol.161, 473–482 (1950)Google Scholar
  2. Baker, R. N., Cancilla, P. A., Pollock, P. S., Frommes, B. S.: The movement of exogenous protein in experimental cerebral edema. J. Neuropathol. Exp. Neurol30, 668–679 (1971)Google Scholar
  3. Bignami, A., Eng, L. F., Uyeda, C. T.: Localization of the glial fibrillary protein in astrocytes by immunofluorescence. Brain Res.43, 429–435 (1972)Google Scholar
  4. Blakemore, W. F.: The fate of escaped plasma protein after thermal necrosis of the rat brain: an electron microscope study. J. Neuropathol. Exp. Neurol.28, 139–152 (1969)Google Scholar
  5. Blinderman, E. E., Markham, Ch. H.: Fluoresein studies of cerebral edema producted by the cryogenic probe. J. Neurosurg.23, 38–44 (1965)Google Scholar
  6. Böhme, G.: Untersuchungen an der area postrema von Gallus domesticus. Acta Neuropathol. (Berl.)21, 308–315 (1972)Google Scholar
  7. Bolton, A. E., Hunter, W. M.: The labelling of proteins to high specific radioactivities by conjugation to a125I-containing alcylating agent. J. Biochem.133, 529–539 (1973)Google Scholar
  8. Brightman, M. W., Klatzo, I., Olsson, Y., Reese, T. S.: The bloodbrain barrier to protein under normal and pathological conditions. J. Neurol. Sci.10, 215–239 (1970)Google Scholar
  9. Broman, T., Radner, S., Svanberg, L.: The duration of experimental disturbances in the cerebrovascular permeability due to circumscribed gross damage of the brain. Acta Psychiat. Neurol.24, 167–173 (1949)Google Scholar
  10. Broman, T., Edström, R., Steinwall, O.: Technical aspects on dyes and radiotracers in the determination of blood-brain barrier damage. Acta Psych. Neurol. Scand.36, 69–75 (1961)Google Scholar
  11. Broman, T., Branemark, P.-I., Johansson, B., Steinwall, O.: Intravital and postmortem studies on air embolism damage of the blood-brain barrier tested with trypan blue. Acta. Neurol. Scand.42, 146–152 (1956)Google Scholar
  12. Bryar, G. E., Goldstein, N. P., Svien, H. J., Sayre, G. P., Jones, J. D.: Experimental cerebral edema: Vital staining with Evans blue during the development and regressive phase. J. Neurosurg.30, 391–398 (1969)Google Scholar
  13. Clasen, R. A., Cooke, P. M., Pandolfi, S., Boyd, D., Raimondi, A. J.: Experimental cerebral edema produced by focal freezing. J. Neuropathol. Exp. Neurol.21, 579–596 (1962)Google Scholar
  14. Clasen, R. A., Cooke, P. M., Pandolf, S., Boyd, D., Raimondi, A. J.: Experimental cerebral edema produced by focal freezing. J. Neuropathol. Exp. Neurol.21, 579–596 (1962)Google Scholar
  15. Clasen, R. A., Cooke, P. M., Pandolf, S., Boyd, D., Raimondi, A. J.: Vital staining mechanisms in the damged and edematous central nervous system. Meeting abstract. 38th Annual Meeting of the American Association of Neuropathologists, Atlantic City, N.J., June 16, 1962. J. Neuropathol. Exp. Neurol.22, 318–363 (1963)Google Scholar
  16. Clasen, R. A., Pandolfi, S., Hass, G. M.: Vital staining serum albumin and the blood-brain barrier. J. Neuropathol. Exp. Neurol.29, 266–284 (1970)Google Scholar
  17. Coons, A. H., Kaplan, M. H.: Localization of antigen in tissue cells. J. Exp. Med.91, 1–13 (1950)Google Scholar
  18. Cutler, R. W., Watters, G. V., Barlow, Ch. W.:125I-labeled protein in experimental brain edema. Arch. Neurol.11, 225–238 (1964)Google Scholar
  19. Edvinsson, L., Nielson, K. C., Owman, Ch., West, K. A.: Alterations in intracranial pressure, blood-brain barrier, and brain edema after sub-chronic implantation of a cannula into the brain of conscious animals. Acta Physiol. Scand.82, 527–531 (1971)Google Scholar
  20. Ehrlich, P.: Das Sauerstoffbedürfnis des Organismus. In: Eine farbenanalytische Studie. Berlin 1885Google Scholar
  21. Esiri, M. M., Taylor, C. R., Mason, D. Y.: Application of an immunoperoxydase method to a study of the central nervous system: preliminary findings in a study of human formalin-fixed material. Neuropath. Appl. Neurobiol.2, 233–246 (1976)Google Scholar
  22. Gregersen, M. I., Rawson, R. A.: The disappearance of T-1824 and structurally related dyes from the blood stream. Am. J. Physiol.138, 698–707 (1943)Google Scholar
  23. Hamberger, A., Hamberger, B.: Uptake of catecholamines and penetration of trypan blue after blood-brain barrier lesions. Z. Zellforschung70, 386–392 (1966)Google Scholar
  24. Hirano, A., Dembitzer, H. M., Becker, N. H., Levine, S., Zimmermann, H. M.: Fine structural alterations of the bloodbrain barrier in experimental allergic encephalomyelitis. J. Neuropathol. Exp. Neurol.29, 432–440 (1970)Google Scholar
  25. Hossmann, K.-A., Olsson, Y.: The effect of transient cerebral ischemia on the vascular permeability to protein tracers. Acta Neuropathol. (Berl.)18, 103–112 (1971)Google Scholar
  26. Hunter, W. M., Greenwood, F. C.: Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature194, 495–496 (1962)Google Scholar
  27. Klatzo, I., Miquel, J.: Observation on pinocytosis in nervous tissue. J. Neuropathol. Exp. Neurol.19, 475–487 (1960)Google Scholar
  28. Klatzo, I.: Presidential address. Neuropathological aspects of brain edema. J. Neuropathol. Exp. Neurol.26, 1–14 (1967)Google Scholar
  29. Klatzo, I.: Application of fluorescence microscopy to the study of the nervous system. In: The central nervous system. International Academy of Pathology Monograph No. 9, p. 319. Baltimore: Williams and Wilkins 1968Google Scholar
  30. Klatzo, I., Piraux, A., Laskowski, E. J.: The relationship between tissue elements, blood-brain barrier and edema in a local brain injury. J. Neuropathol. Exp. Neurol.17, 548–564 (1958)Google Scholar
  31. Klatzo, I., Miquel, J., Otenasek, R.: The application of fluorescein labelled serum protein to the study of vascular permeability in the brain. Acta Neuropathol. (Berl.)2, 144–160 (1962)Google Scholar
  32. Lee, J. C., Olszewski, J.: Effect of air embolism on permeability of cerebral blood vessels. Neurology (Minneap.)9, 614–625 (1959)Google Scholar
  33. Ludwin, S. K., Kosek, J. C., Eng, L. F.: The topographical distribution of S-100 and GFA proteins in the adult rat brain: an immunohistochemical study using horseradish peroxydase-labelled antibodies. J. Comp. Neurol.165, 197–208 (1976)Google Scholar
  34. Lumsden, C. E.: The immunogenesis of the multiple sclerosis plaque. Brain Res.28, 365–390 (1971)Google Scholar
  35. Moriarty, G. C., Moriarty, M. C., Sternberger, L. A.: Ultrastructural immunocytochemistry with unlabelled antibodies and the peroxydase-antiperoxidase complex. J. Histochem. Cytochem.21, 825–833 (1973)Google Scholar
  36. Oldstone, M. B., Dixon, F. J.: Immunohistochemical study of allergic encephalomyelitis. Am. J. Pathol.52, 251–263 (1968)Google Scholar
  37. Olsson, Y., Crowell, R. M., Klatzo, I.: The blood-brain barrier to protein tracers in focal cerebral ischemia and infarction caused by occlusion of the middle cerebral artery. Acta Neuropathol. (Berl.)18, 89–102 (1971)Google Scholar
  38. Rapoport, S. I.: Reversible opening of the blood-brain barrier. Science173, 1026–1028 (1971)Google Scholar
  39. Rawson, R. A.: The binding of T-1824 and structurally related diazo dyes by the plasma proteins. Am. J. Physiol.138, 708–717 (1943)Google Scholar
  40. Reese, T. S., Karnowsky, M. J.: Fine structural localization of a blood-brain to exogenous peroxydase. J. Cell Biol.34, 207–217 (1967)Google Scholar
  41. Rinder, L., Olsson, Y.: Studies on permeability changes in experimental brain concussion. Acta Neuropathol. (Berl.)11, 201–209 (1968)Google Scholar
  42. Tavolato, B. F.: Immunoglobulin G distribution in multiple sclerosis brain. J. Neurol. Sci.24, 1–11 (1975)Google Scholar
  43. Tschirgi, R. D.: Protein complexes and the impermeability of the blood-brain barrier to dyes. Am. J. Physiol.163, 756 (1950)Google Scholar
  44. Westergaard, E., Brightman, M. W.: Transport of proteins across normal cerebral arterioles. J. Comp. Neurol.152, 17–44 (1973)Google Scholar
  45. Woyciechowska, J. L., Brzosko, W. J.: Immunofluorescence study of brain plaques from two patients with multiple sclerosis. Neurology (Minneap.)27, 620–622 (1977)Google Scholar
  46. Vise, W. M., Liss, L., Yashon, D., Hunt, W. E.: Astrocytic processes: a route between vessels and neurons following blood-brain barrier injury. J. Neuropathol. Exp. Neurol.24, 324–334 (1975)Google Scholar
  47. Vulpe, M., Hawkins, A., Rozdilsky, B.: Permeability of cerebral blood vessels in experimental allergic encephalomyelitis studied by radioactive iodinated bovine albumin. Neurology (Minneap.)10, 171–177 (1960)Google Scholar
  48. Weller, T. H., Coons, A. H.: Fluorescent antibody studies with agents of varicella and herpes zoster-propagated in vitro. Proc. Soc. Exp. Biol. Med.86, 789–794 (1954)Google Scholar

Copyright information

© Springer-Verlag 1979

Authors and Affiliations

  • F. Wilmes
    • 1
  • K. -A. Hossmann
    • 1
  1. 1.Forschungsstelle für HirnkreislaufforschungMax-Planck-Institut für HirnforschungKoln 91Federal Republic of Germany

Personalised recommendations