Method for detecting neuronal degeneration and anionic fluorescein homologue stains therefor

• US 6,229,024 B1
• Filed: 01/21/1999
• Issued: 05/08/2001
• Est. Priority Date: 01/23/1998
• Status: Expired due to Term

• Current Assignee: Laurence C. Schmued
• Original Assignee: Laurence C. Schmued
• Inventors: Schmued, Laurence C.
• Primary Examiner(s): Lambkin, Deborah C.
• Application Number:
  US09/234,766
• Time in Patent Office: 838 Days
• Field of Search: 549/391, 436/501
• US Class Current: 549/391
• CPC Class Codes: C07D 311/80 Dibenzopyrans; Hydrogenated dibenzopyrans
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2001/302 Stain compositions
• CPC Class Codes: G01N 33/6896 Neurological disorders, e.g. Alzheimer's disease

Abstract

An anionic fluorescein homologue capable of selectively staining degenerating neurons in brain slices, the method of making the anionic fluorescein homologue, and the method of using the same for detecting neuronal degeneration. Two anionic fluorescein based fluorescent stains are the water soluble tribasic salt of a mixture of the fluorescein isomers 5-carboxyfluorescein and 6-carboxyfluorescein, and the water soluble tetrabasic salt of 5,6 dicarboxyfluorescein.

Claims

1. The method of detecting neuronal degeneration in postmortem brain slices, comprising staining said slices with a fluorescein compound having the formula:

wherein R₁ and R₂ are selected from the group consisting of —

COOX, —

H (provided that both do not=—

H), and

and X is selected from the group consisting of Na⁺, K⁺, Li⁺, and NH₄⁺.

2. A fluorescein compound for use in the detection and localization of degenerating neurons in post-mortem brain slices having the following structure:

wherein R₁ and R₂ are selected from the group consisting of —

COOX, —

H (provided that both do not=—

H), and

and X is selected from the group consisting of Na⁺, K⁺, Li⁺, and NH₄⁺.

3. The method of detecting neuronal degeneration in post-mortem brain slices comprising staining said slices with a fluorescein compound having the following structure:

4. A fluorescein compound for use in the detection and localization of degenerating neurons in post-mortem brain slices comprising a compound having the following structure:

5. The method of detecting neuronal degeneration in post-mortem brain slices comprising staining and slices with a fluorescent compound having the following structure:

6. A fluorescein compound for use in the detection and localization of degenerating neurons in post-mortem brain slices comprising a compound having the following structure:

7. The method of producing the compound:

comprising the steps of;

suspending one gram of 5/6-carboxyfluorescein in 10 ml of distilled water;

adding with stirring 3.4-3.6 ml of 9% NaOH to produce a solution having a final pH between 7.0 and 8.0 with the carboxyfluorescein fully dissolved; and

removing the water to produce deep red crystals with metallic green iridescence.

8. The method of producing the compound:

comprising the steps of;

producing a suspension of 5/6-carboxyfluorescein in distilled water;

adding with stirring an amount of NaOH sufficient to produce a solution having a final pH between 7.0 and 8.0 with the carboxyfluorescein fully dissolved; and

removing the water to produce deep red crystals with metallic green iridescence.

9. The method of claim 8 wherein said water is removed by heating and maintaining said solution at about 40°

C.

10. The method of claim 8 wherein said water is removed by vacuum lyophilization.

11. The method of claim 8 wherein said crystals are pulverized and stored in a desiccator at room temperature.

12. The method of simultaneously producing the following two compounds:

comprising the steps of;

pulverizing and mixing together two moles of resorcinol with one mole of 1,2,4,5-benzenetetracarboxylic dianhydride;

heating the mixture to 180°

C. and maintaining said temperature for two hours;

cooling and pulverizing the fused mass;

mixing said cooled, pulverized mass with distilled water to form a 10% suspension;

adding to said suspension a 9% solution of sodium hydroxide in drop-wise fashion to achieve a pH of about 7.5;

maintaining said solution at room temperature for about 2 hours;

filtering said solution to remove any insoluble materials; and

dehydrating said solution to produce dark red crystals.

13. The method of claim 12 wherein said water is removed by heating and maintaining said solution at about 40°

C.

14. The method of claim 12 wherein said water is removed by vacuum lyophilization.

15. The method of claim 12 wherein said crystals are pulverized and stored in a desiccator at room temperature.

16. The method of detecting neuronal degeneration in post-mortem brain slices, comprising staining said slices with a fluorescein compound having the formula:

wherein one R group consists of COO—

X and the other R group is selected from the group consisting of COO—

X, 3′

hydroxy-6′

ketoxanthenone, and hydrogen, and X is selected from the group consisting of Na⁺, K⁺, Li⁺, and NH₃⁺.

17. A fluorescein compound for use in the detection and localization of degenerating neurons in post-mortem brain slices comprising a fluorescein compound having the structure:

wherein one R group consists of COOX and the other R group is selected from the group consisting of COOX, 3′

hydroxy-6′

ketoxanthenone, and hydrogen, and X is selected from the group consisting of Na⁺, K⁺, Li⁺, and NH₃⁺.