Morphometric assessment of toxicant induced neuronal degeneration in full and restricted contact co-cultures of embryonic cortical rat neurons and astrocytes: using m-Dinitrobezene as a model neurotoxicant.

With m-Dinitrobenzene (m-DNB) as a selected model neurotoxicant, we demonstrate how to assess neurotoxicity, using morphology based measurement of neurite degeneration, in a conventional "full-contact" and a modern "restricted-contact" co-culture of rat cortical neurons and astrocytes. In the "full-contact" co-culture, neurons and astrocytes in complete physical contact are "globally" exposed to m-DNB. A newly emergent "restricted-contact" co-culture is attained with a microfluidic device that polarizes neuron somas and neurites into separate compartments, and the neurite compartment is "selectively" exposed to m-DNB. Morphometric analysis of the neuronal area revealed that m-DNB exposure produced no significant change in mean neuronal cell area in "full-contact" co-cultures, whereas a significant decrease was observed for neuron monocultures. Neurite elaboration into a neurite exclusive compartment in a compartmentalized microfluidic device, for both monocultures (no astrocytes) and "restricted" co-cultures (astrocytes touching neurites), decreased with exposure to increasing concentrations of m-DNB, but the average neurite area was higher in co-cultures. By using co-culture systems that more closely approach biological and architectural complexities, and the directionality of exposure found in the brain, this study provides a methodological foundation for unraveling the role of physical contact between astrocytes and neurons in mitigating the toxic effects of chemicals such as m-DNB.

Assessment of 3-nitropropionic acid-evoked peripheral neuropathy in rats: neuroprotective effects of acetyl-l-carnitine and resveratrol.

Oxidative stress and secondary excitotoxicity, due to cellular energy deficit, are major factors playing roles in 3-nitropropionic acid (3-NPA) induced mitochondrial dysfunction. Acute or chronic exposure to 3-NPA also leads to neuronal degeneration in different brain regions. The present study quantitatively assessed peripheral neuropathy induced by chronic exposure to 3-NPA in rats. The neuroprotective abilities of two antioxidants, acetyl-l-carnitine and resveratrol, were investigated as well. Rats were exposed for up to four weeks to 3-NPA alone or 3-NPA combined with acetyl-l-carnitine or resveratrol, administered peripherally. The experimental outcome was evaluated by neurophysiological, histological, and morphometric analyses. Rats exposed to 3-NPA developed hind limb paresis. Furthermore, a significant decrease in motor nerve conduction velocity (MCV) was detected in tail nerves and axonal degeneration in sciatic nerves (p<0.05). Treatment with resveratrol prevented the functional effects of 3-NPA exposure, whereas treatment with acetyl-l-carnitine, preventing paresis, was not effective to MCV and morphological changes. These data suggest that resveratrol is a good candidate for treatment of metabolic neuropathy. The experimental outcome of this study shows that chronic treatment with 3-NPA in rats is relevant in development of an experimental model of toxic neuropathy.

Quantitative morphological assessment reveals neuronal and glial deficits in hippocampus after a brief subtoxic exposure to chlorpyrifos in neonatal rats.

Neurochemical and behavioral studies indicate that the widely used organophosphorus insecticide, chlorpyrifos (CPF), evokes neurobehavioral teratogenicity with a wide window of vulnerability, ranging from embryonic life through postnatal development. Few studies have detailed morphological damage that corresponds to the operational deficits. We administered 5 mg/kg of CPF sc daily on postnatal days (PN) 11-14, a regimen that is devoid of systemic toxicity, but that elicits long-term cognitive impairment and disruption of cholinergic, catecholaminergic, and serotonergic synaptic function. On PN15 and 20, we conducted quantitative morphologic examinations of neurons and glia in CA1, CA3, and dentate gyrus regions of the hippocampus. Although hippocampal morphology after CPF exposure was normal on gross observation, morphometric analysis revealed a significant overall reduction in the total number of neurons and glia. Superimposed on this basic effect, CPF elicited a delayed-onset increase in the neuron/glia ratio that emerged by PN20, connoting selective gliotoxicity. The alterations in cell numbers were accompanied by significant perikaryal swelling and by enhanced development of astrocytic processes. Layer thickness also showed delayed-onset effects of CPF, with thinning of the CA1 and CA3 layers and enlargement of the dentate gyrus. Our results indicate that there are subtle morphological changes in the juvenile rat brain after neonatal CPF exposure that are detectable only with quantitative analysis and that correlate with regional and cell-specific targets identified earlier in neurochemical studies. The simultaneous targeting of neurons and glia by CPF is likely to play an important role in its developmental neurotoxicant effects.

In vitro and in vivo assessment of the effect of impurities and chirality on methamidophos-induced neuropathy target esterase aging.

In vitro and in vivo studies evaluated neuropathy target esterase (NTE) inhibition and aging (i.e., loss of reactivation potential) by analytical and technical grade racemic and resolved L-(-) and D-(+) isomers of methamidophos (O,S-dimethyl phosphoramidothioate). For studies in vitro, microsomal protein from phenobarbital-induced livers was isolated from chick embryos and NTE inhibition assays were performed using chick embryo brain homogenate treated with 1 or 5 mM methamidophos (with and without metabolic enzymes); for studies in vivo, hens received 30 to 35 mg/kg methamidophos injected into the pectoral muscle. NTE aging in hens was assessed 24 h later or after 30 min to 1 h incubation in vitro using solutions of potassium fluoride (KF) reactivator. Technical methamidophos produced significantly higher levels of aged-inhibited NTE than analytical methamidophos or isolated optical isomers. In vivo, technical methamidophos produced 61% total NTE inhibition with 18% aged and 43% unaged NTE; hens receiving analytical grade averaged 6% aged, 52% unaged, and 58% total NTE inhibition. Results for 1 mM analytical methamidophos in vitro were 5% aged, 54% unaged, and 59% total inhibition; for 1 mM technical methamidophos, values averaged 11% aged, 50% unaged, and 60% total NTE inhibition. The degree of NTE aging obtained both in vivo and in vitro for the isolated D-(+) and L-(-) isomers never exceeded that obtained using analytical grade. These data indicate that impurities in methamidophos could contribute to OPIDN potential. The in vitro methodology described could be applied to first tier screening for detection of NTE inhibition and aging, thus reducing the need for whole-animal testing for OPIDN.

Effects of excitotoxic lesions of the rat ventral striatum on the perception of reward cost.

This study examined the possibility that lesions of the nucleus accumbens in rats impair the perception of the "cost of reward", as defined by the number of operant responses needed to obtain a food pellet. In a first experiment, visual cues indicated the cost of reward under a multiple-ratio schedule of reinforcement. In a second experiment, the number of lever presses required for each reward incremented with each trial in a progressive-ratio schedule of reinforcement. Lesions of the nucleus accumbens altered the behavioral response to the increasing cost of reward when there was an absence of external cues. There was no change in the ability of the lesioned rats to respond to visual cues that indicated reward availability. The results are considered in terms of the traditional idea of the nucleus accumbens as a limbic-motor interface: it is suggested that, if the nucleus accumbens serves such a function, it is limited to only some contexts.