A new murine model of giant proximal axonopathy.

The aromatic gamma-diketone 1,2-diacetylbenzene (1,2-DAB), the putative active metabolite of the organic solvent 1,2-diethylbenzene, forms blue-colored polymeric protein adducts and induces the formation of amyotrophic lateral sclerosis (ALS)-like giant, intraspinal neurofilamentous axonal swellings in Sprague Dawley rats. The pathogenetic mechanism of this neuropathy has yet to be understood. We assessed whether these pathological changes are also seen in the C57BL/6 mouse, the animal of choice for toxicogenomic studies. Mice were treated intraperitoneally with 30, 35, 50, or 70 mg/kg 1,2-DAB or its inactive isomer 1,3-DAB per day (or on alternate days) for up to 43 days. Animals treated with 30 or 35 mg/kg per day 1,2-DAB, but not with 1,3-DAB, developed muscle spasms and progressive weakness, most prominently in hind limbs. Light microscopy revealed swollen axons in spinal anterior horns and proximal ventral roots, and to a lesser extent in dorsal root ganglia of 1,2-DAB-treated animals. Ultrastructural examination of swollen axons revealed clumps of maloriented 10-nm neurofilaments. Sciatic nerves showed clustering of axonal microtubules and other organelles. These findings are qualitatively comparable to those reported in rats treated with 1,2-DAB and represent a suitable phenotype with which to explore molecular mechanisms of proximal, giant neurofilamentous axonopathy using proteomic and genomic technologies.

1,2-diacetylbenzene, the neurotoxic metabolite of a chromogenic aromatic solvent, induces proximal axonopathy.

Several widely used aromatic hydrocarbon solvents reportedly induce blue-green discoloration of tissues and urine in animals and humans. The chomophore has been proposed to result from a ninhydrin-like reaction with amino groups in proteins. The present study examines the neurotoxic property of 1,2-diacetylbenzene (1,2-DAB), the active metabolite of the chromogenic and neurotoxic aromatic solvent 1,2-diethylbenzene. Rats treated with 1,2-DAB, but not with the nonchromogenic isomer 1,3-DAB or with ninhydrin developed blue discoloration of internal organs, including the brain and spinal cord. Only 1,2-DAB induced limb weakness associated with nerve fiber changes, which were most prominent in spinal cord and spinal roots. Changes began with the formation of proximal, neurofilament-filled axonal swellings of the type seen after treatment with 3,4-dimethyl-2,5-hexanedione, a potent derivative of the active metabolite of the neurotoxic aliphatic hydrocarbon solvents n-hexane and methyl n-butyl ketone. These compounds are metabolized to a gamma-diketone that forms pyrroles with target proteins, such as neurofilament proteins. A comparable mechanism is considered for 1,2-DAB, an aromatic gamma-diketone.

Electron microscopic immunolocalization of basic fibroblast growth factor in peripheral nerves.

In spite of ample information about the distribution and the effects of basic fibroblast growth factor (bFGF) in the central nervous system, few data are available concerning the localization of this protein in the peripheral nervous system. In view of the role of bFGF in the regulation of trophic and non-trophic functions, we focused on the presence and precise localization of this growth factor in normal peripheral nerves at the electron microscopic level. The study shows that bFGF is mainly located in the Schwann cells, especially in the nuclei. There is slight labeling in the myelin sheath and in the axon cytoplasm. The study provides morphologic evidence for an association between bFGF expression and Schwann cells. Such as association argues for a role of this peptide in the maintenance or regeneration of peripheral nerves.

Ultrastructural immunolocalization of basic fibroblast growth factor in mast cell secretory granules. Morphological evidence for bfgf release through degranulation.

We previously reported that mast cells (MCs) serve as a source of basic fibroblast growth factor (bFGF), a potent angiogenic and mitogenic polypeptide, suggesting that bFGF may mediate MC-related neovascularization and fibroproliferation. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion, and the mechanism(s) for its release remains controversial. Because MCs release a wide spectrum of bioactive products via degranulation, we hypothesized that MC degranulation may be a mechanism of bFGF release and used ultrastructural immunohistochemistry to test the hypothesis. We reasoned that if bFGF is released through degranulation, it should be localized to MC secretory granules. Human tissues with chronic inflammation and rat/mouse tissues with anaphylaxis were studied. In all tissue samples examined, positive staining (or immunogold particle localization) for bFGF in MCs was predominantly in the cytoplasmic granules. Moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles exhibited minimal immunogold particle localization. In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells. In rat/mouse lingual tissue undergoing anaphylaxis, immunogold particle localization for bFGF was found not only in swollen cytoplasmic granules but also in the extruded granules of MCs. Three different anti-bFGF antibodies gave similar immunogold particle localization patterns, whereas all controls were negative. These results provide morphological evidence suggesting that, despite the lack of a classic secretory peptide in its structure, bFGF is localized to the secretory granules in MCs and may be released through degranulation.

Blister artifact formation due to organic solvent effects on Spurr's epoxy resin semithin sections.

Wrinkles and air bubble artifacts may occur when preparing slides of semithin sections (0.5 microm) from blocks embedded in different resins. More than aesthetically annoying, wrinkles and air bubble artifacts may prohibit study of small structures. Present observations suggest that organic solvent based mounting media may interact with the resin of the section. This sometimes causes wrinkles and air bubble artifacts in the sections that degrade the quality of light microscope images. We compared the quality of semithin sections of several tissues in different resins using various types of mounting media. We observed that sections from Spurr's resin have many more artifacts. In particular, small, 2-10 microm round or oblong blister-like artifacts often plague our Spurr's resin sections. We demonstrate that Spurr's resin sections react with toluene and xylene in organic solvent based mounting media forming blisters, while sections from Araldite and L. R. White do not. We suggest combinations of embedding and mounting media for successful preparation of semithin sections for light microscopy without wrinkles, blisters, or air bubble artifacts.

beta-Cyano-L-alanine toxicity: evidence for the involvement of an excitotoxic mechanism.

The legume Vicia sativa (common vetch) harbors the neurotoxic nonprotein amino acid beta-cyano-L-alanine (BCLA) and its gamma-glutamyl derivative. BCLA elicits hyperexcitability, convulsions, and rigidity in chicks and rats after oral or intraperitoneal administration, but the mechanism of its action is unknown. The effect of different concentrations of BCLA (0.075-10.0 mM) has been investigated in an organotypic tissue culture system. BCLA concentrations of 0.075 and 0.60 mM had no effect, even up to 6 hr. No changes were observed in cultures treated with 1 mM BCLA for 4 hr. BCLA (2.0-10.0 mM) induces concentration-dependent changes in the explants. The explants display neurona vacuolation, chromatin, clumping, and dense shrunken cells, a pathological response generally seen with excitotoxin. MK-801 (35 microM), which blocks the open ion channel associated with the N-methyl-D-aspartate (NMDA) class of glutamate receptors, attenuates the neurotoxic property of BCLA, while the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (10-20 microM), provides no significant protection. Treatment of isolated mouse brain mitochondria with up to 5 mM BCLA had no inhibitory effect on the activity of NADH dehydrogenase (complex I) or cytochrome or oxidase (complex IV), a cyanide-sensitive enzyme. These results suggest that the neurotoxicity of BCLA (or derivative) is mediated directly or indirectly through NMDA receptors.