Adjusting Pediatric Endotracheal Tube Depths Relative to the Cricoid by Using Longitudinal Ultrasound Images of the Saline-Inflated Cuff in the Trachea: Two Case Reports.

Cuff positions of endotracheal tubes should be confirmed to ensure safe anesthesia. However, determining the cuff positions relative to the cricoid by using chest radiography or fiberoptic bronchoscopy is difficult. We identified the cephalad edges of saline-inflated pediatric endotracheal tube cuffs relative to the cricoid on longitudinal ultrasound images over the larynx and trachea in 2 children. Thereafter, we adjusted the endotracheal tube depths and confirmed the cuff positions relative to the cricoid. Longitudinal ultrasound images over the larynx and trachea can help confirm the distance from the caudal edge of the cricoid to the saline-inflated cuff.

Difficulty Inserting Cuffed Endotracheal Tubes in a Child: A Case Report.

We experienced difficulty inserting cuffed inner diameter (ID) 4.5- and 5.0-mm endotracheal tubes (ETTs) in a 5-year-old boy. Postoperative ultrasound investigations showed that the internal transverse width of the cricoid cartilage was 8.0 mm. The maximum outer diameter (OD) of the deflated cuff portion of the cuffed ID 4.5- and 5.0-mm ETTs was 8.5 and 9.6 mm, respectively. The OD of an uncuffed ID 5.5-mm ETT was 7.6 mm; this tube passed the cricoid cartilage. Hence, the transverse width of the cricoid cartilage and ETT diameter including cuff folds should be considered when selecting cuffed ETTs.

Role of endoplasmic reticulum stress in the amygdaloid kindling model of rats.

Endoplasmic reticulum (ER) is an organelle responsible for correct folding and sorting of proteins contributing to neurogenesis and neuronal cell death. We used rapid kindling to analyze specific ER stress marker expression underlying focal epileptogenesis. Seven-week-old rats were divided into three groups: sham (n = 6), partially kindled (n = 8), and over-kindled rats (n = 9). Over kindled rats received over100 stimuli. Partially kindled animals had stimuli halted at stage 2. Protein from ipsilateral hippocampus was electrophoresed on SDS-PAGE, followed by hybridization with primary antibodies, anti-KDEL (-Lys-Asp-Glu-coo-), Bcl-2, BDNF (brain-derived neurotrophic factor), CHOP (C/EBP-homolog protein), C/EBP (CCAAT/enhancer-binding protein), NMDA (N-methyl-D-aspartate; -R1 &2A), GluR1 (glutamate receptor), and ß-tubulin. Western blotting revealed that the ER stress marker BiP (immunoglobulin heavy chain-binding protein) was markedly increased in both partially- and over-kindled groups. BiP expression was ninefold greater than control in partially kindling while twofold greater than control in over-kindled animals. Although ER stress response was accelerated, CHOP expression, which upregulates when apoptosis signaling is accelerated by ER stress, was suppressed. Bcl-2, which acts as an anti-apoptotic molecule, was upregulated in the over-kindled group. Remarkable elevation of BiP was found in partially kindled animals, but not in over-kindled. Over-kindled rats had spontaneous generalized seizure, while partially kindled ones had only partial seizures. Elevation of markers of ER stress in partial seizures might reflect transfer of discharge to contralateral limbic structures. We observed indications of functional changes and neurogenesis in limbic structure during kindling. Widespread indications of functional changes in several membrane and secreted proteins, including NMDA-R1 & R2A and BDNF, for mossy fiber re-construction on the CA3 area, which are related to protein synthesis in the ER, may be important in epileptogenesis.

Differential molecular regulation of glutamate in kindling resistant rats.

Using pentylenetetrazol (PTZ) kindling, we collected hippocampal tissue from standard response and kindling resistant animals, measuring hippocampal mRNA with real-time PCR of glutamate transporters GLAST, GLT-1, and EAAC1 and the sodium-coupled neutral amino acid transporter (SNAT) 1, SNAT2, and SNAT3. In addition, we measured mRNA of glutamine synthetase (GS), phosphate-activated glutaminase (PAG), glutamic acid decarboxylase (GAD) 1, GAD2, and vesicular inhibitory amino acid transporter (VIAAT). Fully kindled animals had decreased expression of mRNA in the hippocampus for GLAST and GAD2 compared with saline injected control. mRNA for SNAT1, SNAT2, SNAT3, GS, and VIAAT was increased. After induction of generalized tonic-clonic seizures by PTZ there were no differences in mRNA at 24h after seizures, equaling baseline quantities except for GAD1, which was decreased. When levels were measured at 30days after a PTZ induced convulsive seizure, we found increased levels of GLT-1, SNAT1 and GS, but decreased levels of GAD1. When these animals, serving as control for the 30day interval between the last convulsive seizure in the kindled experimental group, were analyzed, we found that GLT-1, SNAT3, GAD1 and VIAAT differed in that GLT-1 was decreased and the others increased. Animals found resistant to kindling had strikingly different mRNA patterns, with markedly up-regulated mRNA of proteins that transport glutamate into neurons and glia; SNAT1 was up regulated as well. Up-regulation of genes in kindling resistant animals supports the hypothesis that clearance of glutamate, conversion to glutamine and transport of glutamine into neurons, has the effect of raising the threshold for convulsive seizures and attenuating kindling.

Levetiracetam enhances endogenous antioxidant in the hippocampus of rats: in vivo evaluation by brain microdialysis combined with ESR spectroscopy.

We have attempted to explore the neuroprotective effectiveness of levetiracetam (LEV) by measuring its in vivo antioxidant effect in the hippocampus of rats in a freely moving state. Male Wistar rats were used for the estimation of the in vivo antioxidant effect of LEV through microdialysis combined with electron spin resonance spectroscopy. The antioxidant effect was examined using the principle by which a systemically administered blood-brain barrier-permeable nitroxide radical (PCAM) decreases in an exponential decay manner that is correlated with the amount of antioxidant in the brain. The PCAM decay ratio during perfusion with normal Ringer's solution was compared with that during 32 microM and 100 microM LEV co-perfusion. The in vivo antioxidant effect was examined. In addition, the expressions of the cystine/glutamate exchanger (xCT) and the inducible nitric oxide synthase (iNOS) protein related to redox regulation were measured in the hippocampus of rats after 14 days of administration of LEV at a dose of 54 mg/day i.p. The half-life of PCAM was statistically shortened after LEV perfusion compared with the results of the control experiment. While the expression of the pro-oxidant protein iNOS was decreased, that of the antioxidant protein xCT was statistically increased by the administration of LEV. The role of xCT is to transport cystine, the internal material of glutathione, into the cell. The shortened half-life of the nitroxide radical by co-perfusion of LEV with increased xCT and decreased iNOS expression revealed the enhancement of the endogenous antioxidant effect or free-radical scavenging activity. The results of this study suggest that LEV synergistically enhances the basal endogenous antioxidant effect in the hippocampus with ascorbic acid and alpha-tocopherol. Our findings further suggest that LEV exerts a neuroprotective role by 1) modifying the expression of xCT and iNOS in connection with lipid peroxidation, 2) synergistically enhancing the increased basal endogenous antioxidant ability in the hippocampus, and 3) decreasing the basal concentration of glutamate followed by up-regulation of the intake of cystine, an internal material of GSH.

Role of glutamate and GABA transporters in development of pentylenetetrazol-kindling.

Kindling is a form of epileptogenesis that can be induced with pentylenetetrazol (PTZ). We undertook this study to evaluate the contribution of glutamate and GABA transporters to the process of PTZ kindling. Rats were injected i.p. three times per week with PTZ (40 mg/kg) until they were fully kindled. In rats who achieved full kindling, measurement of hippocampal glutamate and GABA transporters within 24 h by western blot showed that GLAST, GLT-1, and EAAC1 were elevated significantly. However, fully kindled rats at 30 days after their last seizure had no change in either glutamate or GABA transporters proteins. These sequential observations suggest that glutamate transporters may contribute to the occurrence of seizures, but were not associated with maintenance of epileptogenesis. During this experiment, we collected data from animals that had kindled easily and animals who were resistant to kindling. Easily-kindled rats reached full kindling with less than five injections of PTZ. Kindling resistant animals failed to achieve full kindling even after administration of 12 consecutive injections of PTZ. Levels of EAAC1 and GAT-1 in easily-kindled rats were decreased by 30% when compared to kindling resistant animals at 30 days after the last PTZ injection. Since decreased EAAC1 and GAT-1 would diminish GABA function, less quantity of these proteins would appear to be associated with the convulsive threshold at the beginning of kindling development. We wonder if glutamate and GABA transporters might be operant in a convulsion threshold set factor or as a pace factor for kindling.

Molecular regulation of antioxidant ability in the hippocampus of EL mice.

We recently found that the antioxidant ability was remarkably decreased in the hippocampus (Hipp) of EL at 8 weeks of age utilizing ESR spectroscopy. In this study, in addition to evaluating the extracellular glutamate concentration, we tried to determine whether or not changes in the expression of cystine/glutamate exchanger (xCT) and glutamate transporter take place in the Hipp of EL. EL mice and DDY mice at 5, 10, and 20 weeks of age were used for Exp. I and II, respectively. Exp. I: During the interictal state, dialysate was collected from the ventral Hipp using a microdialysis technique, and an extracellular concentration of glutamate ([Glu](o)) was measured with HPLC-ECD. Exp. II: The hippocampal expression of the glutamate transporter and xCT was estimated by Western blots. Exp. I: The level of [Glu](o) at 10 weeks of age was remarkably higher at other ages of EL mice, while [Glu](o) of DDY was unchanged as a result of age. Exp. II: The excitatory amino acid carrier-1 (EAAC-1) and xCT of EL mice at 10 weeks of age decreased more than those of DDY. GLAST and GLT-1 of EL mice at 5 weeks of age decreased more than those of DDY at the same age. No differences were found between EL and DDY for GLAST and GLT-1 at other ages. According to previous studies, the decreased endogenous antioxidant potential observed at 10 weeks of age is a very likely explanation for ictogenesis. The decreased xCT expression at 10 weeks of age could provide the molecular mechanism to explain the depletion of the endogenous antioxidant ability of EL mice during ictogenesis. In addition to the depletion of antioxidant ability, decreased EAAC-1 at this period could be one reason for the collapse of the molecular action of inhibition. These molecular findings support the idea that the elevation of [Glu](o) at 10 weeks of age triggers ictogenesis.

In vivo activation of N-methyl-D-aspartate receptors generates free radicals and reduces antioxidant ability in the rat hippocampus: experimental protocol of in vivo ESR spectroscopy and microdialysis for redox status evaluation.

The pathological mechanisms of various CNS diseases are closely related to glutamate neuronal excitotoxicity following NMDA receptor activation. To verify this relationship, in vivo microdialysis in the hippocampus of rats was applied to ESR spectroscopy during NMDA perfusion. Microdialysis co-perfusion of 0.1 mM NMDA dissolved in 150 mM POBN for 60 min revealed six-line carbon-centered radical ESR spectra. The hfc values were aN=15.7 G and aHbeta=2.5 G, corresponding to the values produced from the generation of lipid radicals. The antioxidant activity during the freely moving state was examined utilizing the principle that systemically applied nitroxide radicals are reduced and lose their paramagnetism by antioxidant activity in the brain. ESR analysis of sequential changes in the signal amplitude of nitroxide radicals in both the NMDA group and the control group revealed an exponential decay. The half-life of the nitroxide radical was significantly longer in the NMDA group than in the control group. The homeostasis of a steady redox balance was destroyed by acute NMDA infusion, which resulted in the generation of lipid radicals and the reduction of antioxidant ability in the hippocampus. The redox imbalance induced by the activation of NMDA-R was recovered by the inhibition of PLA2 and NOS. These results indicated that NMDA-R activation caused the shift to oxidized condition of the redox state, which subsequently leads to neuron death in the hippocampus in the model of glutamate-associated neuronal disease.

In vivo EPR estimation of bilateral hippocampal antioxidant ability of rats with epileptogenesis induced by amygdalar FeCl3 microinjection.

PURPOSE: To measure the neural antioxidant function in the hippocampus of rats with epileptogenesis induced by microinjection of FeCl3 into the amygdala using the decay rate of the nitroxide radical as estimated by L-band electron paramagnetic resonance (EPR) spectroscopy. MATERIALS AND METHODS: Region-selected intensity determination (RSID) was used for the estimation of the nitroxide decay ratio. It is possible to estimate the in vivo hippocampal antioxidant ability using the half-life of the EPR signal of the blood-brain barrier-permeable nitroxide radical. Rats were microinjected with aqueous FeCl3 into the right amygdaloid body. Recording from chronically implanted depth electrodes showed the development of spike discharges with recurrent seizures arising from amygdalar regions with propagation into both hippocampi. Rats with unilateral aqueous FeCl3 lesions were injected systemically with the nitroxide radical and then had EPR for RSID estimation at 5, 15, and 30 days after the iron salt injection. RESULTS: The in vivo antioxidant ability of the dorsal hippocampus was significantly decreased bilaterally in animals with FeCl3-induced seizures when compared to the control. CONCLUSION: Neural antioxidant function in the hippocampi of rats with chronic seizures induced by amygdalar FeCl3 was decreased early and both ipsilaterally and bilaterally.

Age-dependent changes in the hippocampal antioxidant ability of EL mice.

Electron spin resonance (ESR) spectroscopy combined with in vivo microdialysis was used to analyze the antioxidant ability in the hippocampus of mice in an interictal state of EL mice utilizing decay ratio of an exogenously applied nitroxide radical (3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM)). In EL mice with a history of frequent seizures, the half-life of the electron paramagnetism of PCAM in the hippocampus was prolonged. These results revealed decreased antioxidant ability, suggesting vulnerability against oxidative stress. Our data suggest that epileptogenesis in EL mice with chronic seizures is associated with functional failure due to the oxidized redox state and revealed that the decreased hippocampal antioxidant ability is related to the regional vulnerability to oxidative stress in the limbic system of EL mice during epileptogenesis.

Functional role for redox in the epileptogenesis: molecular regulation of glutamate in the hippocampus of FeCl3-induced limbic epilepsy model.

We used western blotting to measure the quantity of glutamate and gamma-aminobutyric acid (GABA) transporters proteins within hippocampal tissue obtained from rats who had undergone epileptogenesis. Chronic seizures were induced by amygdalar injection of FeCl(3). We found that the glial glutamate transporters GLAST and GLT-1 were down-regulated at 60 days after initiation of chronic and recurrent seizures. However, the neuronal glutamate transporter EAAC-1 and the GABA transporter GAT-3 were increased. We performed in vivo microdialysis in freely moving animals to estimate in vivo redox state. We found that the hippocampal tissues were oxidized, resulting in even further impairment of glutamate transport. Our data show that epileptogenesis in rats resulting in chronic and recurrent seizures is associated with collapse of glutamate regulation caused by both the molecular down-regulation of glial glutamate transporters combined with the functional failure due to oxidation.

Effect of levetiracetam on molecular regulation of hippocampal glutamate and GABA transporters in rats with chronic seizures induced by amygdalar FeCl3 injection.

Enhancement of the glutamatergic excitatory synaptic transmission efficacy in the FeCl3 induced epilepsy model is associated with changes in the levels of glutamate and GABA transporter proteins. This study examined the effect of levetiracetam (LEV) on glutamate overflow and glutamate/GABA transporters expression in rats with epileptogenesis induced by the amygdalar injection of 1.0 microl of 100 mM FeCl3 (epileptic rat) and in control rats receiving amygdalar acidic saline injection (non-epileptic rat). In amygdalar acidic saline injected rats, 40 mM KCl-evoked glutamate overflow was significantly suppressed by both 32 and 100 microM LEV co-perfusion. In unilateral amygdalar FeCl3 injected rats, 32 microM LEV was ineffective, but the 100 microM LEV statistically suppressed glutamate overflow. Western blotting was employed to determine the hippocampal expression of glutamate/GABA transporters in epileptic or non-epileptic rats. The rats were treated for 14 days with 54 mg/kg LEV or vehicle intraperitoneally injection. Following 14 days of treatment, the ipsilateral hippocampus was removed for a Western blot analysis. In non-epileptic rats, the expression increased for all of the glutamate and GABA transporters (GLAST, GLT-1, EAAC-1, GAT-1 and GAT-3) while the glutamate transporter regulating protein (GTRAP3-18) decreased in comparison to those of normal rats that were treated with the vehicle. In epileptic rats receiving LEV, the EAAC-1 and GAT-3 levels increased while GTRAP3-18 (89%) decreased in comparison to those of the epileptic rats treated with the vehicle. GTRAP3-18 inhibitor regulates glutamate-binding affinity to EAAC-1. The anti-epileptic action of LEV may be partially due to a reduction of glutamate-induced excitotoxicity and an enhancement of the GABAergic inhibition as observed with the inhibitory effect on the 40 mM KCl-evoked glutamate overflow. These conclusions are supported by the increase in the expression of glial glutamate transporters (GLAST and GLT-1), and the increase in the expression of EAAC-1 and GAT-3 associated with a decrease in GTRAP3-18. The increased expression of EAAC-1 and the decreased expression of GTRAP3-18 in association with the up-regulation of GAT-3 due to such continual LEV administration was thus found to enhance GABA synthesis and reverse the transport of GABA both in non-epileptic and epileptic rats. The suppression of glutamate excitation and the enhancement of GABA inhibition in the rats with continual LEV administration is a result of the up-regulation of glutamate and GABA transporters with the down-regulation of GTRAP3-18. These observations together demonstrated the critical molecular mechanism of the anti-epileptic activity of LEV.

Protective role of pentobarbital pretreatment for NMDA-R activated lipid peroxidation is derived from the synergistic effect on endogenous anti-oxidant in the hippocampus of rats.

We have attempted to explore the neuroprotective effectiveness of PBT by measuring anti-oxidant ability in the hippocampus of rats in a freely moving state. Anti-oxidant ability was examined utilizing the principle that blood-brain barrier-permeable nitroxide radicals (PCAM) injected i.p. lose their paramagnetism in an exponential decay correlated with anti-oxidant ability in the brain. The half-life of PCAM was used as the indicator of the hippocampal anti-oxidant ability. While the half-life was statistically prolonged when infused with 0.1mM NMDA without PBT, the half-life was almost the same as in the control when infused with NMDA under anesthesia with PBT. In addition, the half-life under only PBT anesthesia was the shortest of all the groups. Our findings, therefore, suggested that PBT anesthesia not only suppresses NMDA-R activated free radical generation but also synergistically enhances the increased basal endogenous anti-oxidant ability in the hippocampus.

An acute dysfunction of the glutamate transport activity has been shown to generate free radicals and suppress the anti-oxidant ability in the hippocampus of rats.

In this study, we attempted to elucidate whether or not an acute inhibition of glutamate transports activity with l-trans-pyrrolidine-2,4-dicarboxylic acid (l-trans PDC) would cause neuroexcitoxicity in the hippocampus. We used in vivo microdialysis and X-band electron spin resonance (ESR) spectroscopy to measure the changes in the redox state during the perfusion of l-trans PDC. ESR signals from rats using l-trans PDC were characteristically a six-line spectra, for which the hfc was a(N)=1.57mT and a(H)=0.25mT; these hfc's were obtained from the lipoxygenase/linoleic acid system that was used for the generation of lipid radicals. The antioxidant effect was measured using an ESR analysis to monitor sequential changes in the signal amplitude of nitroxide radical in the dialysate of both l-trans PDC and control animals. The pattern showed exponential decay with median half-life of the nitroxide radical took significantly longer in the l-trans PDC group. Acute changes in the glutamate transport resulted in the generation of a lipid radical and a depletion in the anti-oxidant effect in the hippocampus. Our data indicate that a dysfunction of a glutamate transport resulted in the collapse of the redox state, which thus eventually led to neuronal necrosis in the hippocampus. This study provides clear evidence for the mechanisms associated with neuronal disorder in relation to glutamate.

Functional role of GABA transporters for kindling development in GLAST KO mice.

Kindling-induced after discharge in electroencephalograms depends on the protein associated with glutamatergic and/or GABAergic neuronal transmission. In glutamate transporter knockout (GLAST KO) mice, the kindling phenomena in GLAST KO developed more slowly while the after discharge duration (ADD) was briefer than that of the control C57BL-6J mice. These findings indicate that either the excitatory function was suppressed or the inhibitory function was enhanced in GLAST KO kindling. To explain these phenomena, we used Western blotting to evaluate the alterations in the expression of hippocampal GABA transporter proteins, and the estimation of the effect on the process of epileptogenesis. Although no alterations were observed in the GAT-3 expression, the hippocampal GAT-1 expression was significantly suppressed in comparison to that of C57BL-6J mice. A decreased GAT-1 level in the hippocampus, which might be associated with the increased extracellular GABA level, may therefore inhibit both ADD and seizure propagation as shown by the amygdaloid kindling phenomenon observed in GLAST KO mice.

Generation of lipid radicals in the hippocampus of neonatal rats after acute hypoxic-ischemic brain damage.

Free radical-mediated lipid peroxidation has been strongly suggested to be the main cause of neuronal toxicity in the rat brain, including neonatal brain damage. The primary objective of this experiment was to see if the generation of free radicals occurred in the acute phase of ischemic-hypoxic insult in neonatal rats, by electron paramagnetic resonance (EPR) spectroscopy and in vivo brain microdialysis. A spin trap agent, alpha-(4-pyridyl-N-oxide)-N-tert-butylnitrone was perfused through a probe in the hippocampus before and after hypoxia and then an analysis was performed by EPR. From the EPR analysis of spin adduct in the dialysates, we obtained the EPR spectrum of six line spectra for which the hyperfine coupling constants corresponded to those of the EPR signal from the lipoxygenase/linoleic acid (LPX/LA), a lipid radical generating system, increased transiently just after hypoxia. The results of our in vivo study show the lipid peroxidation of the neuronal membrane to progress during neonatal ischemic-hypoxic insult. We hypothesize that an increased formation of lipid radicals may participate in the cascade of reactions leading to neuronal damage in the hippocampus following ischemic-hypoxic insult in neonatal rats.

Molecular regulation of glutamate and GABA transporter proteins by clobazam during epileptogenesis in Fe(+++)-induced epileptic rats.

To assess the molecular effects of the antiepileptic drug clobazam (CLB, 1,5-benzodiazepine), a benzodiazepine effective in the management of epilepsy, we performed a series of experiments using rats with chronic, spontaneous recurrent seizures induced by amygdalar injection of FeCl(3). Experimental animals were treated for 14 days with CLB. We then measured the expression of glutamate and GABA transporter proteins and evaluated the changes that occurred in these proteins using both experimental and control animals. CLB treatment was associated with an increase in the production of GLT-1 in the contra-lateral hippocampus of animals receiving amygdalar FeCl(3) and CLB treatment. CLB treatment up-regulated the GABA transporter GAT3 in the contra-lateral hippocampus of animals with chronic, recurrent seizures. In contrast, CLB had no effect on the expression of EAAC1 and GAT1 in the hippocampus or the cortex in control animal groups. Chronic epileptogenesis may be associated with down-regulation of the production of glial excitatory amino acid transporters, GLAST and GLT-1, proteins that cause increase in the basal extracellular concentrations of glutamate. Elevated GABA transporter expression results in increased reverse transport of GABA to the extracellular space during periods of excitation. In addition to allosteric activation of GABA(A) receptors, this study suggests that CLB might exhibit its antiepileptic action by increasing GLT-1 expression and GAT3 in the hippocampus of rats with chronic seizures.

In vivo evaluation of the effect of zonisamide on the hippocampal redox state during kainic acid-induced seizure status in rats.

The aim in this study is to observe the hippocampal redox state during kainic-acid (KA)-induced seizure status, and examine the effect of systemic preinjection of anticonvulsant zonisamide (ZNS) on the hippocampal redox. To perform under a freely moving state, in vivo microdialysis method was applied to electron paramagnetic resonance (EPR) spectroscopy. Half-life of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM), a five-membered ring nitroxide radical, was used for the indicator of the hippocampal antioxidant ability. The changes in EPR signal intensities of PCAM decreased exponentially in all rats used. The average half-lives of PCAM was significantly shorter in the rats pretreated with ZNS than that of control group, and while the average half-lives of PCAM in the perfusate was significantly longer in the rats KA-induced status epilepticus than that of control (P < 0.01). Those of PCAM in the ZNS-pretreated rats followed by KA-injection were almost the same as those of control. These findings indicate that the pretreatment of ZNS increased the antioxidant ability in the hippocampus during KA-induced seizure. This study is the first in vivo evaluation of the antioxidant ability of ZNS as neuroprotective role against the free radicals performed under the condition of freely moving rats during seizure status.

Kinetics of extracellular nitroxide radical and glutamate levels in the hippocampus of conscious rats: cautionary note to the application of nitroxide radical on clinical arena.

Recently, novel applications of the nitroxide radicals have been proposed as antioxidant and anti-cancer agents. In view of the significance of nitroxide radical as a potential pharmaceutical agent for various applications in biological systems, it will be important to investigate further whether nitroxide radicals have a neurotoxicity or not. Blood-brain barrier permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM; five-membered ring nitroxide radical) would provide us more important information to explore the neuronal excitotoxicity of nitroxide radicals on the central nervous system. Every rat injected with PCAM showed limbic seizure with secondary generalization. PCAM administration resulted in neuronal cell loss in CA1 area, which is closely associated with the neurotoxicity of endogenous glutamate and nitroxide itself. More detailed studies on their possible toxicity of nitroxide radicals will be needed before the prospect of moving nitroxide from the experimental to the clinical arena when nitroxide radicals would be used for CNS disease in future.