Hajime Nakanishi

The Neuroprotective Effect of the Novel Noncompetitive NMDA Antagonist, FR115427 in Focal Cerebral Ischemia in Rats

The present study was carried out to compare the neuroprotective effect of the novel noncompetitive NMDA antagonist, FR115427, with that of (+)MK-801 in rat focal cerebral ischemia. Focal cerebral ischemia was produced by permanent occlusion of the left middle cerebral artery (MCA). Drugs were administered intraperitoneally immediately after ischemia and once a day for 6 successive days. FR115427 (10 mg/kg, i.p.) significantly improved neurologic deficit at 1 day after ischemia and reduced total infarct volume (54%) at 7 days after ischemia. Although FR115427 (10 mg/kg, s.c.) produced neuronal vacuolization similar to (+)MK-801, FR115427 did not produce adverse effects such as a loss of body weight, mortality, and hypothermia, in contrast to (+)MK-801. These results suggest that FR115427 may be useful in the treatment of stroke.

A colorimetric assay method for the evaluation of neurotrophic activity in vitro.

A colorimetric assay was established to detect neurotrophic activity by measuring the lysosomal enzyme, acid phosphatase (AP) activity of cultured neuronal cells. Neurons from the cerebral cortex of 14- or 15-day mouse embryo were cultured in serum-free medium for 3 days in 96-well culture plates. A linear relationship was obtained between the AP activity and the number of viable neurons counted under a microscope. The AP assay was used to evaluate the neurotrophic activity of basic fibroblast growth factor. This assay is shown to be simple, sensitive and convenient to detect neurotrophic activity.

Neuronal survival and neurite extension supported by astrocytes co-cultured in transwells

The influence of astrocytes on the development of cerebral cortical neurons was studied in a co-culture system using transwells with chemically defined medium. Cerebral cortical neurons from 15- or 16-day-old mouse embryo were cultured in the lower wells which were separated by a porous membrane from the upper transwells where cerebral cortical astrocytes from newborn mouse were cultured. Neurons co-cultured with astrocytes for 7 days formed a network-like web and maintained a slightly better survival from 4 to 7 days. However, neurons cultured in conditioned medium obtained from astrocytes did not form any network after 7 days even though they maintained a better cell survival at 4 days.

Influence of Glucose Supply and Demand on Determination of Brain Glucose Content with Labeled Methylglucose

The equilibrium brain/plasma distribution ratio for 3-O-methyl-D-glucose (methylglucose) varies with plasma and tissue glucose contents and can be used to determine local glucose levels in brain. This ratio was previously found to rise as brain glucose concentration fell in response to lowered plasma glucose content. The ratios, however, differed with the same tissue glucose levels in conscious and pentobarbital-sedated rats, suggesting that changes in metabolic demand might alter the quantitative relationship between the methylglucose distribution ratio and brain glucose concentration. To examine this possibility, metabolic rate was varied by focal drug application, and hexose concentrations measured in treated and surrounding tissue. When tissue glucose levels were reduced by raised metabolic demand, methylglucose distribution ratios also fell. When brain glucose levels rose due to reduced consumption, the methylglucose distribution ratio also rose. Thus, in contrast to the inverse relationship between brain/plasma methylglucose ratio and brain glucose concentration when brain glucose content is altered secondarily to changes in plasma glucose level, changes in brain glucose content induced by altered glucose utilization cause the brain glucose level and methylglucose distribution ratio to rise and fall in a direct relationship. Determination of brain glucose content from methylglucose distribution ratios must take into account rates of glucose delivery and consumption.

Neurotrophic effect of isoquinoline derivatives in primary cortical culture

Recent studies indicate that the N-methyl-D-aspartate (NMDA) antagonist, (+)-1-methyl-1-phenyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (FR 115427), enhanced neuronal survival in primary culture of cortical neurons from mouse embryos. In the present study isoquinoline derivatives were examined for the neurotrophic activity in primary culture of cortical neurons and were also examined for anti-NMDA activity. In spite of varying level of anti-NMDA activity, isoquinoline derivatives enhanced neuronal survival at the concentration of 10 microM. To elucidate of the mechanisms of neurotrophic activity in primary cortical culture, nicardipine and flunarizine, known calcium channel blockers, were also tested. Neither nicardipine nor flunarizine showed neurotrophic activity up to the doses causing toxicity in cultured neurons. NBQX, an AMPA receptor antagonist, was also tested for neurotrophic activity. However no enhancement of neuronal survival was observed. These data suggest that one of the mechanisms to promote neuronal survival may depend on the structure of isoquinoline ring. Moreover neurotrophic activity observed in our culture systems might not relate on anti-NMDA activity, blockade of voltage dependent L-type calcium channels and antagonization of AMPA receptor.

Non-competitive NMDA antagonists, FR115427 and MK801, enhance neuronal survival in primary culture

Primary culture of cerebral cortical neurons from mouse embryo was treated with non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, FR115427 and MK801. Both FR115427 and MK801 at 1-10 microM promoted neuronal survival after 7 days cultivation, determined by calorimetric assay for acid phosphatase (AP) activity. FR115426, the (-) isomer of FR115427 also promoted neuronal survival. However, competitive NMDA antagonists, APH and CGS19755 did not show promotive effect on neuronal survival at up to 100 microM. Anti-NMDA activity of these compounds was evaluated using an NMDA-induced convulsion assay. The relative potency of anti-NMDA activity was CGS19755 > MK801 > APH > FR115427 > FR115426. These data suggest that the effects on neuronal survival by FR115427 and MK801 are independent of anti-NMDA activity.