The use of rat brain slices as an in vitro model for mechanistic evaluation of neurotoxicity-studies with acrylamide

Biochemical mechanisms underlying acrylamide induced neurotoxicity were examined using an in vitro model consisting of sagittal slices of rat brain. Incubation of brain slices under oxygen in artificial cerebrospinal fluid containing acrylamide produced a dose and time dependent inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Lysosomal enzymes, acid phosphatase, N-acetyl glucosaminidase and beta-glucuronidase decreased in a similar manner, while no changes were observed in the activity of Na+K+ATPase, cytochrome c oxidase and lactate dehydrogenase. Incubation of slices with two structurally related compounds, acetamide (a non-neurotoxic amide) and methylene bis-acrylamide (a weak neurotoxin), indicated that acrylamide selectively inhibited GAPDH, enolase and N-acetyl glucosaminidase at low concentration; similar doses of acetamide and methylene bis-acrylamide did not have the same effect on brain slices. Incubation with acrylamide depleted glutathione levels in slices, and the addition of glutathione to the incubation medium prevented acrylamide induced inhibition of GAPDH and lysosomal enzymes. Time dependent inhibition of lysosomal enzymes was also observed in vivo, in the brain and sciatic nerve of rats following a single dose of acrylamide. These results demonstrate that both in vitro and in vivo, lysosomal enzymes are also inhibited following acrylamide exposure. The rat brain slice model exhibits both selectivity and sensitivity towards neurotoxicants and hence, may prove to be an useful in vitro model for the mechanistic evaluation of neurotoxicity.     

Opposite effects of phospholipase A2 on [3H]AMPA binding in adult and neonatal membranes.

In the present study, we compared the effect of phospholipase A2 (PLA2) treatment of synaptic membranes from adult and neonatal rats on the characteristics of [3H]AMPA binding sites. Whereas PLA2 treatment of membranes from adult rats produces an increased affinity for [3H]AMPA binding, the same treatment in neonatal rats results in a decrease in the maximal number of binding sites. Since activation of PLA2 has been proposed to play a critical role in the formation of long-term potentiation (LTP), possibly mediated through a modification of the AMPA receptors, the results strengthen the hypothesis that PLA2-induced modification of [3H]AMPA binding sites is an important component of synaptic plasticity.     

Differential effects of specific opioid receptor agonists on rat pup isolation calls.

The possibility was investigated that specific opioid receptor types might selectively alter the production of isolation-induced ultrasonic vocalizations. Intracisternal injections of mu, delta and kappa opioid receptor agonists were administered to isolated 10-day-old rat pups. The mu receptor agonist [D-Ala2-NMe-Phe4-Gly-ol]-enkephalin (DAMGO) and delta receptor agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) both reduced the rate of isolation-induced ultrasonic calling in the absence of sedation. The kappa receptor agonist U50,488 had the opposite effect, significantly raising the rate of vocalization. Fourteen-day-old pups, with a larger delta receptor population, showed a greater sensitivity to DPDPE than was seen in the younger animals.     

Methylmercury induced alterations in the nerve growth factor level in the developing brain.

Pre- and early postnatal stages in the development of the central nervous system (CNS) are very sensitive to the toxic effects of methylmercury. The influence of methylmercury on the level of nerve growth factor (NGF) during the development of CNS was studied. Sprague-Dawley rats were exposed indirectly throughout the fetal and suckling periods until weaning on postnatal day 25 (P 25) via their dams given methylmercury in the diet (3.9 mg/kg diet). In addition, after weaning offsprings were exposed directly to methylmercury via the diet until postnatal day 50 (P 50). The level of NGF was analyzed in cortical areas and in the septum with a sensitive enzyme immunoassay. The pups exposed to MeHg exhibited a 50% elevation in the level of NGF in the hippocampus on P 25 and P 50 compared to control animals. Concomitantly, the level of NGF decreased by 30% in the septum on P 25 and P 50, suggesting that the retrograde transport of NGF from hippocampus to septum could be affected by the exposure of methylmercury. The exact mechanism by which the low level of mercury is affecting the NGF concentration in the developing brain is yet unknown. The increase of NGF in the hippocampus and the decrease of NGF measured in the septum could reflect altered conditions for neurotrophic support in these areas of the brain as a result of the exposure to heavy metal. Thus, this finding might indicate a connection between exposure of heavy metals and neurodegeneration, such as that found in the basal forebrain in Alzheimer's disease.     

Chimeric cytotoxin IL2-PE40 inhibits relapsing experimental allergic encephalomyelitis.

IL2-PE40 is a chimeric protein composed of human interleukin-2 (IL2) genetically fused to a modified form of Pseudomonas exotoxin lacking the cell recognition domain. IL2-PE40 is cytotoxic for IL2 receptor-bearing lymphocytes in culture and can inhibit activation of T cells in vivo. IL2-PE40 can significantly diminish antigen-stimulated proliferation of lymphocytes sensitized to myelin basic protein. Intraperitoneal administration of IL2-PE40 not only markedly inhibits the clinical manifestations of adoptively transferred relapsing experimental allergic encephalomyelitis but also dramatically reduces both inflammation and demyelination characteristic of the disease.     

Tetrahydroaminoacridine improves passive avoidance retention defects induced by aging and medial septal lesion but not by fimbria-fornix lesion.

The present study examines whether tetrahydroaminoacridine (THA) can improve the deterioration in passive avoidance (PA) retention performance induced by medial septal (MS) and fimbria-fornix (FF) lesions in young rats or by aging. Retention of young MS-lesioned rats was improved by pretraining injection of THA at 3 mg/kg, but not by THA at 1 mg/kg or by either of the posttraining doses of THA (1 and 3 mg/kg). Pretraining injections of THA at 1 or 3 mg/kg had no effect on the PA retention performance of FF-lesioned rats. Age-induced PA failure was alleviated by pretraining administration of THA at 1 and 3 mg/kg. Posttraining injections of THA (1 or 3 mg/kg) had no effect on PA retention performance of aged rats. These results demonstrate that 1) THA may improve hippocampal cholinergic denervation-induced functional deficits and 2) some of the age-related PA deficits may be due to a cholinergic deficit and can be reversed with THA.     

Does chronic prenatal methadone exposure affect beta-receptor subtypes in placental, fetal and maternal brain homogenates?

Computer competition analysis of 3H-DHA (3H-dihydroalprenolol, a nonselective beta-adrenergic radioligand) binding in the presence of unlabeled metoprolol (a beta 1-selective antagonist) indicates the existence of both beta 1- and beta 2-adrenergic receptor subtypes in the rat placenta and confirms previous reports that both beta-adrenoceptors are present in adult rat cortex. In the fetal brain (20th day of gestation), however, only beta 1-receptors were detected. Pregnant rats were chronically exposed to methadone from day 7 to day 20 of gestation via implanted osmotic minipumps (6.3-9.0 mg/kg/day). This treatment schedule did not induce a change in the affinity and density of either beta-receptor subtype in the placental, fetal and maternal brain homogenates. The results are discussed in terms of the reported monoaminergic and opiate receptor functional interactions.     

Therapy of human cervical carcinoma with monoclonal antibody-Pseudomonas exotoxin conjugates.

Pseudomonas exotoxin A (PE) linked to the F(ab')2 fragment of 1H10, a murine monoclonal antibody recognizing a carbohydrate epitope of a glycoconjugate expressed on the surface of human cervical carcinoma tumor cells, was evaluated for in vitro and in vivo activity. PE can kill cells by ADP-ribosylating elongation factor 2 thus inhibiting protein synthesis. Disulfide- as well as thioether-linked immunotoxins (1H10-PE) killed cervical carcinoma cells in vitro and were 20-160 times more inhibitory to target than to control cells. Cell killing was antibody mediated as demonstrated by the reduction of 1H10-PE growth inhibition to target CaSki cells by free 1H10 F(ab')2. In addition, a control antibody immunotoxin was nontoxic to CaSki cells. Thioether-linked 1H10-PE administered either i.v. or i.p. suppressed the growth of established solid s.c. cervical carcinoma tumors xenografted in nude mice for over 30 days. Treatment with antibody alone or a control immunotoxin had no significant effect on tumor growth. Administration of immunotoxin i.p. was associated with less toxicity than administration i.v., but i.v. injections were more effective at suppressing the growth of established solid tumors.