Background: Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved.Results: Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H₂O₂) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H₂O₂. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H₂O₂-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H₂O₂-treated cells.Conclusions: In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells.

A functional interaction between the corticotropin-releasing factor (CRF) system and noradrenergic neurons in the brain has been suggested. In the present study, we investigated the interrelationship between the central CRF-induced elevation of plasma catecholamines and adrenoceptor activation in the paraventricular nucleus of the hypothalamus (PVN) using urethane-anesthetized rats. In rats under urethane anesthesia, a femoral venous line was inserted for infusion of saline, and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for the application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed with high-performance liquid chromatography with electrochemical detection. Quantification of noradrenaline in rat PVN microdialysates was performed with high-performance liquid chromatography with electrochemical detection. We showed that centrally administered CRF elevated noradrenaline release in the PVN. Furthermore, we demonstrated that microinjection of phenylephrine into the PVN induced elevation of plasma levels of adrenaline, but not of noradrenaline, whereas microinjection of isoproterenol into the PVN induced elevation of plasma levels of noradrenaline, but not of adrenaline. Bilateral blockade of adrenoceptors in the PVN revealed that phentolamine significantly suppressed the CRF-induced elevation of plasma adrenaline level, while propranolol significantly CRF-induced elevation of plasma noradrenaline level. Our results suggest that centrally administered CRF-induced elevation of plasma levels of adrenaline and noradrenaline can be mediated via activation of α-adrenoceptors and β-adrenoceptors, respectively, in the rat PVN.

Domoic acid (DA) is an excitatory amino acid analogue of kainic acid (KA) that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS) to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo.

Acute restraint stress (RS) induces sympathetic activation such as elevating plasma catecholamines, resulting in increase in blood glucose. We aimed to investigate whether glucose infusion affects the RS-induced sympathetic responses. Plasma catecholamines were measured by high-performance liquid chromatography with electrochemical detection. Blood glucose levels were measured with a glucometer and a glucose assay kit. Cardiac parameters were measured by echocardiographic and hemodynamic analysis. Prostanoid levels in the paraventricular nucleus of hypothalamus (PVN) microdialysates were measured by liquid chromatography-ion trap tandem mass spectrometry analysis. RS significantly increased plasma noradrenaline and adrenaline. Intravenous infusion of a 5% glucose solution significantly attenuated the RS-induced elevation of plasma adrenaline but did not alter the plasma noradrenaline. Glucose administration during RS suppressed the progression of cardiac impairment by attenuating the decline rates in left ventricular diastolic, end-diastolic volume, stroke volume, fractional shortening, and ejection fraction. Both Intravenous and intracerebroventricular infusion of glucose solution significantly attenuated the RS-induced elevation of thromboxane B₂ (TxB₂) (a metabolite of TxA₂) levels in the PVN but did not alter prostaglandin E₂ levels in the PVN. Our results demonstrate that glucose infusion suppresses RS-induced elevation of plasma adrenaline and left ventricular dysfunction. In the brain, glucose infusion suppresses RS-induced production of TxA₂ in the PVN.