Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1999-01-21
2001-05-08
Lambkin, Deborah C. (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C436S501000
Reexamination Certificate
active
06229024
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for the post-mortem detection and localization of degenerating neurons in brain slices, and more particularly to anionic fluorescent type homologue therefor.
2. Prior Art
Many neuroscientists including toxicologists, pathologists, pharmacologists, anatomists and histochemists share an interest in detecting neuronal degeneration. The demonstration of this phenomenon, however, is often difficult from both a technical and an interpretational point of view. Conventional techniques such as H&E (hematoxylin and eosin), or Nissl type stains (crystal violet, thionin, etc.) are technically simple and can be used to infer degeneration based on changes such as neuronal shrinkage, vacuolation, and hyperchromatism. Chassan, J. L., “Nervous System” in W. A. D. Anderson and J. M. Kissane (Eds.),
Pathology,
Mosby, St. Louis, 1977, pp. 2074-2148; Garcia, J. H. and Kamijiyo, Y., “Cerebral Infraction: Evolution of Histopathological Changes After Occlusion of a Middle Cerebral Artery in Primates,” J. Neuropathol. Exp. Neurol., 33 (1974) pp. 409-421; and Siesjo, B. K., “Cell Damage in the Brain: a Speculative Synthesis,” Cereb. Blood Metabol., 1 (1981) 155-185. Unfortunately, such changes are not necessarily indicative of neuronal degeneration and may be due to processing artifacts or non-lethal alterations in cellular morphology. Processing artifacts can result in both shrunken and hyperchromatic cells. Cammermeyer. J., “The Importance of Avoiding the “dark” Neurons in Experimental Neuropathology,” Acta. Neuropath. 1 (1961) 245-270; and Stensaas, S. S., Edwards, C. Q., and Stensaas, L. J., “An Experimental Study of Hyperchromic Nerve Cells in the Cerebral Cortex,” Exp. Neurol., 36 (1972) 472-487. Not only are such techniques prone to false positives, but it is also possible to miss degenerating neurons since all cells stain with these dyes while only relatively subtle morphological differences exist between normal and degenerating neurons. These interpretational difficulties make the analysis of conventionally stained material time consuming. In contrast, suppressed silver techniques are much better from this point of view in that normal neurons remain unstained while degenerating neurons stain black. de Olmos, J. S., Beltramino, C. A., and de Olmos de Lorenzo, S. “Use of an amino-cupric-silver technique for the detection of early and simiacute neuronal degeneration caused by neurotoxicants, hypoxia, and physical trauma,” Neurotox.& Teratol., 16, (1994) 545-561; Fink, R. P., Heimer, L., “Two methods for the selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system,” Brain Res., 4, (1967) 369-374; Gallyas, F., Wolf J. R., Bottcher, H., and Zaborsky, L., “A reliable and sensitive method to localize terminal degeneration and lysosomes in the central nervous system,” Stain Technol., 55 (1980) 299-306; and Nauta, W. J. H. and Gygax, P. A., “Silver impregnation of degenerating axons in the central nervous system: A modified technique,” Stain Technol., 29 (1954) 91-93. The main drawbacks associated with suppressed silver techniques lie in their labor intensive and capricious nature.
OBJECTS OF THE INVENTION
The principal object of the present invention is to provide an improved histochemical tracer which is simple and reliable to use as well as highly sensitive and easy to interpret.
Another object of the present invention is to provide an improved histochemical tracer of the foregoing character which is a sensitive and definitive marker of neuronal degeneration.
A further object of the present invention is to provide an improved histochemical tracer of the foregoing character which is simple to use and produces reliable results.
SUMMARY OF THE INVENTION
The present invention is embodied in an anionic fluorochrome capable of selectively staining degenerating neurons in brain slices, the method of making the anionic fluorochrome, and the method of using the same for detecting neuronal degeneration. The histochemical application of this fluorochrome results in a simple, sensitive and reliable method for staining degenerating neurons and their processes. The technique will detect neuronal degeneration resulting from exposure to a variety of neurotoxic insults. The fluorochrome can be combined with other fluorescent methodologies including immunofluorescence, fluorescent axonal tract tracing, and fluorescent Nissl counterstaining. Compared to conventional methodologies, the fluorochrome of the present invention is a more sensitive and definitive marker of neuronal degeneration than conventional H&E or Nissl type stains, while being comparably sensitive yet considerably simpler and more reliable than suppressed silver techniques.
The novel anionic fluorochorme of the present invention is more specifically a tri- or tetra-salt, such as a sodium salt of a 5/6-carboxy fluorescein. The fluorochrome may for example be trisodium 5/6-carboxy fluorescein or tetrasodium 5/6-carboxy fluorescein.
REFERENCES:
patent: 4508892 (1985-04-01), Yoshida
patent: 4615986 (1986-10-01), Yoshida
patent: 5623080 (1997-04-01), Neckers et al.
patent: 6008379 (1999-12-01), Benson et al.
Ali, S.F., Duhart, H.M., Newport, G.D., Lipe, G.W., and Slikker, W. Jr., Manganese-induced reactive oxygen species: Comparison between Mn+2and Mn+3, Neurodegen., 4, (1995) 329-334.
Auer, R.N., Olson, Y., and Siesjo, B.K., Hypoglycemic brain injury in the rat. Correlation of density of brain damage with the EEG isoelectric time: a quantitative study, Diabetes, 33 (1984) 109-1098.
Binienda, Z., Frederick, D.L., Ferguson, S.A., Rountree, R.L., Paule, M.G., Schmued, L.C., Ali, S.F., Slikker, W. Jr., and Scallet, A.C., The effect of perinatal Hypoxia on the behavioral, neurochemical, and neurohistological toxicity of the metabolic inhibitor 3-nitropropionic acid, Metabolic Brain Disease, 10/4 (1995) 269-281.
Cammermeyer, J., The Importance of avoiding the “dark” neurons in experimental Neuropathology, Acta. Neuropath., 1 (1961) 245-270.
Chassan, J.L., Nervous System. in W.A.D. Anderson and J.M. Kissane (Eds.), Pathology, Mosby, St. Louis, 1977, pp. 2074-2148.
deOlmos, J.S., Beltramino, C.A., and de Olmos de Lorenzo, S. Use of an amino-cupric-silver technique for the detection of early and simiacute neuronal degeneration caused by neurotoxicants, hypoxia, and physical trauma, Neurotox.& Teratol., 16, (1994) 545-561.
Desai, V.G., Feuers, R.J., Hart, R.W., and Ali, S.F., MPP+induced neurotoxicity in mouse is age-dependent: Evidenced by the selective inhibition of complexes of electron transport, Brain Res., 715 (1996) 1-8.
Donaldson, J., McGregor, D., and La Bella, F., Manganese neurotoxicity: a model for free radical mediated neurodegeneration? Can. J. Physiol. Pharmacol., 60 (1982), 1398-1405.
Fink, R.P., Heimer, L., Two methods for the selective silver impregnation of degenerating axons and their synaptic endings in the centralq nervous system, Brain Res., 4, (1967) 369-374.
Fix, A.S., Horn, J.W., Wrightman, K.A., Johnson, C.A., Long, G.G., Storts, R.W., Faber, N., Wozniak, D.F., and Olney, J.W., Neuronal vacuolization and necrosis induced by the non-competitive N-methyl-D-aspartate (NMDA) antagonist MK (+) 801 (dizocilpine maleate): a light and electron microscopic evaluation of the rat retrosplenial cortex, Exp. Neurol., 123 (1993) 204-215.
Gallyas, F., Wolf J.R., Bottcher, H., and Zaborsky, L., A reliable and sensitive method to localize terminal degeneration and lysosomes in the central nervous system, Stain Technol., 55 (1980) 299-306.
Garcia, J.H. and Kamijiyo, Y., Cerebral infraction: Evolution of histopathological changes after occlusion of a middle cerebral artery in primates, J. Neuropathol. Exp. Neurol., 33 (1974) pp. 409-421.
Gary, L.J. and Powell, T.P.S., The projections of the retina in the cat, J. Anat. (Lond.) 102 (1968) 189-222.
Hamilton, B.F. and Gould, D.H., Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic: a type of hypoxic (energ
Crandell Ralph F.
Lambkin Deborah C.
LandOfFree
Method for detecting neuronal degeneration and anionic... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for detecting neuronal degeneration and anionic..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for detecting neuronal degeneration and anionic... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2483832