Summary
A direct fluorescence-microscopic technique was effected to determine in the central nervous system (CNS) of the mouse the distribution of ethidium bromide after intravenous (i.v.) injection. The compound was visualized in thin cryostat sections of the brain fixed by vascular perfusion through the heart with a 10% buffered formalin solution.
Ethidium bromide emitted a bright red fluorescent light in model experiments. The compound could not be detected in the vessel walls or brain parenchyma of the cerebral gray and white matters after i.v. injection indicating the presence of a blood-brain barrier (BBB) phenomenon to this compound.
Signs of extravasation of ethidium bromide were present in the choroid plexus, the postremal area, the Gasserian ganglion, and in the circumventricular organs of the brain (neurohypophysis, organum vasculosum lamina terminalis, and median eminence) 3 min after the i.v. injection. Intense fluorescence was present in the nucleus and the cytoplasm of the cells in these areas, located outside of the BBB. Fluorescence had disappeared 24h after the injection. Unexpectedly, red fluorescent material was seen in the parenchyma of the olfactory lobes of some animals, indicating, possibly, the presence of ethidium bromide.
Ethidium bromide is known to suppress RNA, DNA, and protein synthesis in mammalian cells and has been used previously in neuropathology for studies on myelin lesions after injury to oligodendroglial cells. It can now, by a simple fluorescence-microscopic method, be traced directly in fixed tissue. Correlations can therefore be made between localization of the compound and its cytotoxic effects. For instance, the influence of a primary injury to the protein synthesizing organelles in cells of the circumventricular organs can be explored after i.v. injection of ethidium bromide. As the compound does not seem to pass the BBB, selective lesions to the nervous system might be produced by direct microinjections or possibly by the use of retrograde axonal transport mechanisms. It would be essential in such experiments to ascertain in which cells the compound was located, and fluorescence-microscopic technique presented here may be useful for this purpose.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arvidson B (1979) Distribution of intravenously injected protein tracers in peripheral ganglia of adult mice. Exp Neurol 63:388–410
Bengtsson A, Grimelius L, Johansson H, Pontén J (1977) Nuclear DNA-conent of parathyroid cells in adenomas, hyperplastic and normal glands. Acta Pathol Microbiol Scand [A] 85:455–460
Bigotte L, Arvidson B, Olsson Y (1982) Cytofluorescence localization of adriamycin in the nervous system. 1. Distribution of the drug in the central nervous system of normal adult mice after intravenous injection. Acta Neuropathol (Berl) 57:121–129
Bigotte L, Olsson Y (1982a) Cytofluorescence localization of adriamycin in the nervous system. III. Distribution of the drug in the brain of normal adult mice after intraventricular and arachnoidal injections. Acta Neuropathol (Berl) 58:193–202
Bigotte L, Olsson Y (1982b) Retrograde transport of doxorubicin (adriamycin) in peripheral nerves of mice. Neurosci Lett 32:217–221
Bigotte de Almeida L (1983) Cytological localization and toxicity of adrimycin in the nervous system. Acta Univ Upsaliensis 465:1–61
Bigotte L, Olsson Y (1983) Cytotoxic effects of adriamycin on mouse hypoglossal neurons following retrograde axonal transport from the tongue. Acta Neuropathol (Berl) 61:161–168
Blakemore WF (1982) Ethidium bromide-induced demyelination in the spinal cord of the cat. Neuropathol Appl Neurobiol 8:365–375
Bouchard C (1975) Données ultrastructurales sur la perméabilité des capillaires des organes circumventriculaires du cerveau. J Microse Biol Cell 24:45–58
Bradbury M (1979) The concept of a blood-brain barrier. Wiley, New York
Bradley WG, Krasin F (1982) DNA hypothesis of amyotrophic lateral sclerosis. Adv Neurol 36:439–500
Broadwell RD, Brightman MW (1976) Entry of peroxidase into neurons of the central and peripheral nervous systems from extracerebral and cerebral blood. J Comp Neurol 166:257–284
Browlee G, Gors MD, Goodwin LG, Woodbine M, Wall LP (1950) The chemotherapeutic action of phenanthridine compounds. Br J Pharmacol 5:261–275
Cesarini K, Bigotte L, Olsson Y (1984) Fluorescence-microscopic localization of in vivo injected ethidium bromide in the nervous system of the mouse. Acta Neuropathol (Berl) 63:78–79
Cozzarelli NR (1977) The mechanism of action of inhibition of DNA synthesis. Ann Rev Biochem 46:64–668
Genest D, Wahl Ph, Erard M, Champagne M, Daune M (1982) Fluorescence anisotrophy decay of ethidium bromide bound to nucleosomal core particles. Biochimie 64:419–427
Jacobs JM, MacFarlane RM, Cavanagh JB (1976) Vascular leakage in the dorsal root ganglia of the rat, studies with horseradish peroxidase. J Neurol Sci 29:95–107
Koda LY, van der Kooy D (1983) Doxorubicin: a fluorescent neurotoxin retrogradely transported in the central nervous system. Neurosci Lett 36:1–8
Le Pecq JB, Paoletti C (1967) A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol 27:87–106
Mann DMA, Yates PO (1982) Ageing, nucleic acids and pigments. Rec Adv Neuropathol 2:109–138
Olsson Y (1984) Vascular permeability in the peripheral nervous system. In: Dyck PJ, Thomas PK, Lambert EH, Bunge R (eds) Peripheral neuropathy, 2nd edn. Saunders, Philadelphia, pp 579–598
Plumbridge TW, Brown JF (1977) Spectrophotometric and fluorescence polarization studies of the binding of ethidium, daunomycin and mepacrine to DNA and to poly (IC). Biochim Biophys Acta 479:441–449
Rapoport SI (1976) Blood-brain barrier in physiology and medicine. Raven Press, New York
Reese TS, Karnovsky MJ (1967) Fine structural localization of a blood brain barrier to exogenous peroxidase. J Cell Biol 34:207–217
Slonimski PP, Perrodin G, Groft JH (1968) Ethidium bromide-induced mutation of yeast mitochondria: Complete transformation of cells into respiratory. Deficient nonchromosoma “petites”. Biochem Biophys Commun 30: 232–240
van Deurs B (1980) Structural aspects of brain barriers with special reference to the permeability of the cerebral endothelium and choroidal epithelium. Int Rev Cytol 65:117–140
Waring MJ (1981) DNA modification of cancer. Ann Rev Biochem 50:159–192
Woolfe G (1952) The trypanocidal action of phenanthridine compounds. Ann Trop Med Parasitol 46:285–293
Yajima K, Suzuki K (1979a) Ultrastructural changes of oligodendroglia and myelin sheaths induced by ethidium bromide. Neuropathol Appl Neurobiol 5:49–62
Yajima K, Suzuki K (1979b) Demyelination and remyelination in the rat central nervous system following ethidium bromide injection. Lab Invest 41:385–392
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Cesarini, K., Atillo, A., Bigotte, L. et al. Cytofluorescence localization of ethidium bromide in the nervous system of the mouse. Acta Neuropathol 68, 273–278 (1985). https://doi.org/10.1007/BF00690829
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00690829