The neuroprotective effect of ONO-1714 on NMDA-mediated cytotoxicity in vitro.

We report the effects of a newly developed NOS inhibitor on the neurotoxicity induced by NMDA on cultured fetal rat cortical neurons. To date, three different isoforms of NOS have been characterized. It has been considered that both neuronal NOS and inducible NOS activities are detrimental to the ischemic brain, whereas endothelial NOS plays a prominent role in maintaining cerebral blood flow and prevents neuronal injury during ischemia. ONO-1714 is a newly developed competitive NOS inhibitor that has selective inhibitory potency for iNOS than eNOS. However, its effect on nNOS has not been investigated yet. In this study, we investigated the neuroprotective effect of ONO-1714 on NMDA-induced neurotoxicity in our established model of primary cultured cortical neurons of rat foetus. Cortical neurons (prepared from E16 rat foetuses) were used after 13-14 days in culture. The cells were exposed to 30 muM NMDA for 24 h in the culture. To evaluate the neuroprotective effects of NOS inhibitors, ONO-1714 and L-NAME, neurons were exposed to various concentrations of an NOS inhibitor with 30 muM NMDA. The NMDA induced neurotoxicity was significantly attenuated by ONO-1714 in all concentrations, but not in low to moderate concentrations of L-NAME. These findings demonstrate that the neuroprotective effect of ONO-1714 was more potent than L-NAME. Moreover, ONO-1714 has a strong inhibitory effect on nNOS and would be a powerful tool for the protection of neurons against cerebral ischemia.

Effects of deep hypothermia on nitric oxide-induced cytotoxicity in primary cultures of cortical neurons.

Nitric oxide (NO) is thought to play a major role during cerebral ischemia. However, the protective efficacy of hypothermia against NO-induced neurotoxicity remains to be examined. In the present study, the degree of neurotoxicity induced by NO was analyzed in two temperature groups (normothermia, 37 degrees C; deep hypothermia, 22 degrees C) of cultured E16 Wistar rat cortical neurons. Two different NO donors, 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC-12) and 1-hydroxy-2-oxo-3-(3-amynopropyl)-3-isopropyl-1-triazene (NOC-5), that have equal half-lives at 37 degrees C and 22 degrees C, respectively, were used. Cultured neurons in each temperature group were exposed to 30 and 100 micro M NOC for three different time courses, 6 hr, 12 hr, and 24 hr. The survival rates of neurons were evaluated by assessing viable neurons on photomicrographs before and after the experiments. The highest survival rate (approximately 93%) was seen in both temperature groups when neurons were exposed to 30 micro M NOC for 6 hr and 12 hr, and there was no significant difference observed between these two groups (P > 0.05). Almost equal survival rates were observed in both temperature groups following exposure to 30 micro M NOC for 24 hr (at 37 degrees C, 80.4% +/- 2.6%; at 22 degrees C, 83.2% +/- 1.6%; P > 0.05). During exposure to 100 micro M NOC, although the survival rate linearly decreased (approximately from 70% to 5%) in both temperature groups when exposed for 6-24 hr, there were no significant intergroup differences observed (P > 0.05). In conclusion, hypothermia does not provide adequate protection to the neurons by acting on the mechanisms evoked by NO, so we speculate that hypothermia may not confer neuroprotetcion once NO is released during ischemia.

Hypothermia and thiopentone sodium: individual and combined neuroprotective effects on cortical cultures exposed to prolonged hypoxic episodes.

Because there are many conflicting reports on cerebroprotective effects of hypothermia and barbiturates, we examined the degree of neuroprotection at defined temperatures (normothermia, 37 degrees C; mild hypothermia, 32 degrees C; deep hypothermia, 22 degrees C; and profound hypothermia, 17 degrees C) and various concentrations (low, 4 microM; moderate, 40 microM; and high, 400 & microM) of thiopentone sodium (TPS), alone and in combination in cortical cultures exposed to prolonged hypoxia (24-48 hr). The survival rate of embryonic day (E)16 Wistar rat cortical neurons was evaluated on photomicrographs before and after experiments. During the 24-hr hypoxic period, the survival rate of neurons was maximal with combinations of mild hypothermia with 40 microM (91.6 +/- 0.7%) and 400 microM TPS (90.8 +/- 0.7%) or deep hypothermia combined with all concentrations of TPS (4 microM, 90.6 +/- 1.0%; 40 microM, 91.4 +/- 0.8%; 400 microM, 91.8 +/- 1.2%). During 48 hr hypoxia, the highest survival rate was seen with the combination of deep hypothermia and either 40 microM (90.9 +/- 0.6%) or 400 microM (91.1 +/- 1.4%) TPS. In the presence of profound hypothermia in combination with all concentrations of TPS, the survival rate was significantly reduced (P< 0.01) compared to combined application of either mild or deep hypothermia with TPS. In summary, maximal neuroprotection was attained with hypothermia and TPS in combination rather than applied individually, during prolonged hypoxic episodes (24- 48 hr). During a 24-hr hypoxic period, both mild and deep hypothermia combined with a clinically relevant concentration of TPS (40 microM) offered the highest neuroprotection. Only deep hypothermia provided maximal neuroprotection when combined with 40 microM TPS, during 48-hr hypoxia. Combination of profound hypothermia and TPS did not confer considerable neuroprotection during long lasting hypoxia.