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.

Ultrastructural immunolocalization of basic fibroblast growth factor in fibroblasts and extracellular matrix.

Basic fibroblast growth factor (bFGF) is thought to play an important role in normal tissue repair and wound healing. It is a potent mitogenic and chemotactic factor for fibroblasts, regulating proliferation and extracellular matrix (ECM) production by these cells. In this study, we present morphologic evidence of the ultrastructural location of bFGF in fibroblasts and ECM using several antibodies, tissues, and species. Distinct labeling is seen in the nuclei of fibroblasts and some labeling in the cytosol. Immunolabeling of the cytosol excludes organelles involved in the usual secretory pathway, such as rough endoplasmic reticulum, Golgi apparatus, and secretory vacuoles. The same labeling is observed with either polyclonal or monoclonal antibodies. We suggest that bFGF functions as a nuclear protein in fibroblasts and is not secreted by a normal secretory pathway. Fibroblasts may export bFGF via unique cellular pathways that are clearly distinct from classic signal peptide mediated secretion. This may provide a source for ECM-resident bFGF. The same antibodies show different labeling intensity in the ECM. This protein, through integration into the ECM, may act as a local regulator and promote regeneration of these tissues after wounding. Direct evidence is the dramatic reduction of bFGF labeling in axotomized rat ECM collagen fibers versus control animals.

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.

Comparison of elemental concentrations in the acinar cells of the human labial salivary gland.

Two types of acinar cells were observed in human labial glands by conventional and analytical electron microscopic and light microscopic techniques. The predominant type contained large and prominent secretory granules that were strongly mucicarmine and PAS (with and without diastase) positive. The second type contained small, lacy, secretory granules, and these cells were faintly positive with these stains. The elemental contents of the two types of granules were measured by analytical electron microscopy using digital mapping and spot analysis applied to freeze-dried cryosections prepared from gland slices incubated in vitro under non-stimulated conditions. The large secretory granules had significantly higher Ca, S and Mg concentrations and significantly lower Cl and K concentrations than the small granules. The difference in elemental contents probably reflects differences in the content of secretory macromolecules. Specifically, the S content is thought to reflect the anionic properties of the secretory macromolecules, while the levels of divalent cations are thought to be determined by electroneutrality requirements for macromolecular folding and storage. No differences were found in nuclear or cytoplasmic elemental concentrations between the two cell types.