L-ephedrine-induced neurodegeneration in the parietal cortex and thalamus of the rat is dependent on hyperthermia and can be altered by the process of in vivo brain microdialysis.

Multiple doses of the dietary supplement L-ephedrine can cause severe hyperthermia and modest dopamine depletions in the rat brain. Since D-amphetamine treatment can result in neurodegeneration, the potential of L-ephedrine to produce similar types of degeneration was investigated. Adult male rats, some implanted in the caudate/putamen (CPu) for microdialysis, were given four doses of 25 mg/kg L-ephedrine or 5 mg/kg D-amphetamine (2 h between doses) at an ambient temperature of 23 degrees C. L-ephedrine-induced degeneration in the forebrain was dependent on the degree of hyperthermia. Layer IV of the parietal cortex was the most sensitive to L-ephedrine treatment with peak body temperatures of at most 40.0 degrees C necessary to produce degeneration. Extensive neurodegeneration in the parietal cortex after L-ephedrine treatment was as pronounced as that previously described for D-amphetamine treatment and also occurred in the intralaminar, ventromedial and ventrolateral thalamic nuclei in rats with severe hyperthermia (peak body temperatures>41.0 degrees C). The neurodegeneration induced by L-ephedrine may have resulted in part from excitotoxic mechanisms involving the indirect pathways of the basal ganglia and related areas. No differences were observed between microdialysis and non-implanted rats with respect to degree of tyrosine hydroxylase (TH) loss in the CPu after either D-amphetamine or L-ephedrine treatment. However, neurodegeneration resulting from D-amphetamine and L-ephedrine was reduced in the microdialysis animals in the hemisphere ipsilateral to the probe, which raises concerns when using the technique of in vivo microdialysis to evaluate neurodegeneration. The results of this study, in conjunction with human clinical evaluation of ephedrine neurotoxicity, indicate that regionally specific damage may occur in the cortex of some humans exposed to ephedrine in the absence of stroke or hemorrhage.

Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration.

Fluoro-Jade B, like its predecessor Fluoro-Jade, is an anionic fluorescein derivative useful for the histological staining of neurons undergoing degeneration. However, Fluoro-Jade B has an even greater specific affinity for degenerating neurons. This notion is supported by the conspicuous staining of degenerating neuronal elements with minimal background staining. This improved signal-to-noise ratio means that fine neuronal processes including distal dendrites, axons and axon terminals can be more readily detected and documented. Although the staining time and dye concentration are reduced, the method is as rapid, simple and reliable as the original Fluoro-Jade technique. Like Fluoro-Jade, Fluoro-Jade B is compatible with a number of other labeling procedures including immunofluorescent and fluorescent Nissl techniques.

Temporal progression of kainic acid induced neuronal and myelin degeneration in the rat forebrain.

The excitatory amino acid glutamate has been implicated in the neurodegeneration associated with several different central nervous system diseases. Treatment with kainic acid (KA), a glutamate analog known to activate the AMPA/KA subtype of glutamate receptor, has been widely used as a model of epilepsy. Long term temporal studies of its neuropathological effects, however, are lacking. In this study, two techniques were used to directly visualize and characterize the neuropathology that occurred over a 2-month period following KA-induced status epilepticus in adult female Sprague-Dawley rats. Post-injection survival was 2, 4, 8 h, 2 days, 2 weeks, or 2 months. Labeling with Fluoro-Jade B (FJB), a fluorescent green dye that labels the cell body, dendrites, axons and axon terminals of degenerating neurons, was observed within the cortex, hippocampus, thalamus, basal ganglia, and amygdala by 4 h post-treatment. The highest level of labeling was seen in the piriform cortex, hippocampus, and thalamus. Myelin changes in the rat forebrain following KA treatment were also examined using the myelin-specific Black-Gold (BG) stain. Varicose myelinated fibers were observed in the same regions as FJB positive neurons, although these changes were evident by the 2-h survival time-point. Both stains showed a temporal progression of brain damage throughout the affected areas. By 2 months post-treatment, few degenerating neurons could be detected and abnormal myelin was absent in most regions. As myelin changes can be seen prior to neuronal degeneration, and oligodendrocytes express functional AMPA/kainate-type glutamate receptors, the neurodegeneration and myelin pathologies may occur as independent events. Thus, researchers should consider the temporal and multiple effects of kainic acid to optimize conditions for their endpoint of interest when designing experiments.

Fluoro-Jade: novel fluorochromes for detecting toxicant-induced neuronal degeneration.

Two anionic fluorescein derivatives can be used for the simple and definitive localization of neuronal degeneration in brain tissue sections. Initial work on the first generation fluorochrome, Fluoro-Jade, demonstrated the utility of this compound for the detection of neuronal degeneration induced by a variety of well-characterized neurotoxicants, including kainic acid, 3-nitropropionic acid, isoniazid, ibogaine, domoic acid, and dizocilpine maleate (MK-801). After validation, the tracer was used to reveal previously unreported sites of neuronal degeneration associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), methamphetamine, and d-fenfluramine. Preliminary findings with a second generation fluorescein derivative, Fluoro-Jade B, suggest that this tracer results in staining of optimal contrast and resolution in animals dosed with kainic acid. These 2 tracers can be combined with other histologic methods, including immunofluoresence and fluorescent Nissl stains. Recent preliminary findings on a number of specialized applications of Fluoro-Jade include the detection of apoptosis, amyloid plaques, astrocytes, and dead cells in tissue culture.

Characterization of N-methyl-d-aspartate receptors in the hyperammonemic sparse fur mouse.

The N-methyl-d-aspartate (NMDA) receptor, a glutamate receptor subtype, is a ligand-gated ion channel. Overstimulation of NMDA receptors may increase intracellular Ca2+ concentrations to lethal levels in neurodegenerative disorders affecting the basal ganglia. Such excitotoxicity may also contribute to the loss of medium spiny neurons in the striata of the hyperammonemic sparse fur (spf/Y) mouse, a model of the X-linked disorder of the urea cycle, ornithine carbamoyltransferase deficiency (OCTD). Levels of quinolinic acid (QA), a potent NMDA agonist, are elevated in the brains of spf/Y mice. Further, direct injection of QA into the striatum produces selective degeneration of medium spiny neurons. Microglia, an endogenous source of QA in the brain, are abundant in spf/Y mice during the period of neuronal degeneration. The location and density of NMDA receptors was visualized by gold labelled immunocytochemistry with a polyclonal antibody to the NMDAR1 receptor subtype and their distribution quantified. A 58% reduction was found in the median density value in the layer V pyramidal neurons in fronto-parietal cortex (p<0.001), but no significant change was observed in the striatum. NMDA receptor binding was examined using [3H]dizocilpine ([3H]MK-801). Receptor density (Bmax) in the striata of clinically stable spf/Y mice and +/Y littermates was unchanged, but was decreased 15% (p<0.01) in the fronto-parietal cortices in clinically stable spf/Y mice compared with +/Y littermate controls.

Dendritic alterations in cortical pyramidal cells in the sparse fur mouse.

Ornithine carbamoyltransferase deficiency, an X-linked trait, leads to toxic hyperammonemia in sparse fur (spf/Y) mice. Quantitative analysis of the basilar dendritic tree of layer V pyramidal cells in frontoparietal cortex stained by the Golgi Kopsch method revealed a significant decrease in both the complexity of the dendritic arbor and in dendritic terminal spine density (60%) in spf/Y mice compared with controls. Such reductions may contribute to behavioral dysfunction observed in spf/Y mice.

Cimetidine-nicoumalone interaction in man: stereochemical considerations.

1. The cimetidine-nicoumalone interaction was studied in five subjects who received a single 10 mg oral dose of racemic nicoumalone alone and 3 days into an oral regimen of cimetidine of 200 mg three times daily and 400 mg at night. 2. The concentrations of R(+)- and S(-)-nicoumalone in plasma were measured using a stereospecific h.p.l.c assay; augmentation of prothrombin time was used as a measure of response. 3. Cimetidine increased the rate (but not extent) of absorption of both R(+)- and S(-)-nicoumalone, and reduced the clearance of R(+)-nicoumalone but not that of the S(-)-enantiomer. 4. Cimetidine increased the anticoagulant response produced by nicoumalone in some but not all subjects, despite a consistent effect on the pharmacokinetics of the oral anti-coagulant. 5. Cimetidine appears to produce its effect by stereoselectively inhibiting the elimination of R(+)-nicoumalone.

Rate and extent of absorption of clonidine from a transdermal therapeutic system.

The in-vivo performance of a clonidine transdermal therapeutic system (TTS 3.5 cm2, 2.5 mg) was assessed in 12 healthy normal volunteers. Particular attention was paid to the rate and extent of absorption of clonidine from the TTS dosage form by reference to a 2 h i.v. infusion of clonidine. The absolute bioavailability of clonidine from the TTS dosage form was found to be approximately 60% with clonidine being released from the TTS at a relatively reproducible and consistent rate of 4.32 micrograms h-1 over a 7-day period.

Stereospecific assay of nicoumalone: application to pharmacokinetic studies in man.

1. A stereospecific h.p.l.c. assay of nicoumalone in plasma has been developed. 2. The assay was applied to a study in which 20 mg racemic nicoumalone was given orally to three volunteers and blood samples taken for 168 h. 3. The mean pharmacokinetic parameters of the individual enantiomers were: clearance/bioavailability 1.28 1 h-1, R-enantiomer; 17.5 1 h-1, S-enantiomer: volume of distribution/bioavailability 12.5 1, R-enantiomer; 22.6 1, S-enantiomer: terminal half-life 6.8 h, R-enantiomer; 0.91 h, S-enantiomer. 4. The data are consistent with a substantial first-pass hepatic loss of S-nicoumalone.

Enoxacin-warfarin interaction: pharmacokinetic and stereochemical aspects.

The interaction between the new quinoline-azaquinoline antibiotic enoxacin and the oral anticoagulant warfarin was investigated in six healthy male volunteers. Enoxacin was found not to affect the hypoprothrombinemic response produced by warfarin but did produce a decrease in the clearance of the less pharmacologically potent enantiomer of warfarin, (R)-warfarin. The decreased clearance of (R)-warfarin produced by concomitant enoxacin administration was found to be a consequence of inhibition by enoxacin of the (R)-6-hydroxywarfarin metabolic pathway.

Pharmacokinetics of imipenem and cilastatin after their simultaneous administration to the elderly.

The pharmacokinetics of imipenem and cilastatin were studied in a group of six healthy elderly male volunteers following the combined intravenous administration of 500 mg imipenem and 500 mg cilastatin sodium as either single or multiple (6 hourly for 6 days) 20 min constant-rate infusions. The pharmacokinetics of both imipenem and cilastatin in the elderly were similar to those of young individuals with mild renal failure (Verpooten et al., 1984). There was no change in the pharmacokinetics of either species with time following multiple-dosing. Correlations existed between total clearance and the glomerular filtration-rate (51Cr-EDTA) for both imipenem and cilastatin.

Comparative effects of ranitidine and cimetidine on the pharmacokinetics and pharmacodynamics of warfarin in man.

Stereochemical aspects of the potential interaction between the oral anticoagulant warfarin and the H2-antagonists, cimetidine and ranitidine, were investigated. A single 25 mg oral dose of racemic warfarin was administered on Day 4 of a randomised 9-day multiple dosing regimen of either cimetidine (800 mg o.d.) ranitidine (300 mg o.d.) or placebo. The degree of anticoagulation produced by warfarin was quantificated by the determination of both the prothrombin and Factor VII clotting times. Ranitidine had no effect on the pharmacodynamics of warfarin or the pharmacokinetics of the individual warfarin enantiomers. Cimetidine whilst producing no statistically significant change in the pharmacodynamics of warfarin or in the pharmacokinetics of the pharmacologically more potent (S) enantiomer, did produce a statistically significant decrease in the clearance of the (R) enantiomer, possibly due to metabolic inhibition of this species.