Anatoly E. Martynyuk

Bumetanide Alleviates Epileptogenic and Neurotoxic Effects of Sevoflurane in Neonatal Rat Brain

Background:We tested the hypothesis that in newborn rats, sevoflurane may cause seizures, neurotoxicity, and impairment in synaptic plasticity—effects that may be diminished by the Na+–K+–2Cl− cotransporter 1 inhibitor, bumetanide. Methods:Electroencephalography, activated caspase-3, and hippocampal long-term potentiation were measured in rats exposed to 2.1% sevoflurane for 0.5–6 h at postnatal days 4–17 (P4–P17). Results:Arterial blood gas samples drawn at a sevoflurane concentration of 2.1% showed no evidence of either hypoxia or hypoventilation in spontaneously breathing rats. Higher doses of sevoflurane (e.g., 2.9%) caused respiratory depression. During anesthesia maintenance, the electroencephalography exhibited distinctive episodes of epileptic seizures in 40% of P4–P8 rats. Such seizure-like activity was not detected during anesthesia maintenance in P10–P17 rats. Emergence from 3 h of anesthesia with sevoflurane resulted in tonic/clonic seizures in some P10–P17 rats but not in P4–P8 rats. Bumetanide (5 &mgr;mol/kg, intraperitoneally) significantly decreased seizures in P4–P9 rats but did not affect the emergence seizures in P10–P17 rats. Anesthesia of P4 rats with sevoflurane for 6 h caused a significant increase in activated caspase-3 and impairment of long-term potentiation induction measured at 1 and 14–17 days after exposure to sevoflurane, respectively. Pretreatment of P4 rats with bumetanide nearly abolished the increase in activated caspase-3 but did not alleviate impairment of long-term potentiation. Conclusion:These results support the possibility that excitatory output of sevoflurane-potentiated &ggr;-aminobutyric acid type A/glycine systems may contribute to epileptogenic and neurotoxic effects in early postnatal rats.

Ionic basis of the differential effects of Intravenous anesthetics on erythromycin-induced prolongation of ventricular repolarization in the guinea pig heart

Background: Dysrhythmias and death occur in patients with acquired long QT syndrome (LQTS). Little information exists regarding interactions between anesthetics and drugs that prolong ventricular repolarization. Therefore the effects of three commonly used intravenous anesthetics on ventricular repolarization were investigated in the setting of drug‐induced, long QT syndrome. Methods: The effects of increasing concentrations (0, 10, 25, and 50 micro Meter) of propofol, ketamine, and thiopental on ventricular repolarization were evaluated by measuring the monophasic action potential duration at 90% repolarization (MAPD90) in guinea pig Langendorff‐perfused hearts in the absence or presence of erythromycin (100 micro Meter). If an anesthetic enhanced erythromycin‐induced prolongation of MAPD90, its effects on the delayed rectifier (IK) and inward rectifier (IKl) potassium currents were measured using the whole‐cell patch‐clamp technique. Results: At clinically relevant concentrations, only thiopental significantly modulated erythromycins effect on MAPD90. Thiopental at 10, 25, and 50 micro Meterr prolonged MAPD90 from a control of 163 +/‐ 6 ms by 18 +/‐ 4, 30 +/‐ 3, and 31 +/‐ 4 ms, respectively. In a separate group, erythromycin prolonged MAPD90 from 155 +/‐ 2 ms to 171 +/‐ 2 ms (n = 21, P < 0.001). In the presence of erythromycin, thiopental at 10, 25, and 50 micro Meter caused significantly greater prolongation from a control of 171 +/‐ 2 ms by 39 +/‐ 2, 58 +/‐ 3, and 72 +/‐ 6 ms, respectively. Whole‐cell patch‐clamp experiments indicated that thiopental inhibited IK and IKl. Conclusions: Intravenous anesthetics caused markedly different effects on ventricular repolarization. Thiopental, unlike propofol and ketamine, potentiated the effects of erythromycin on ventricular repolarization by inhibiting IK and IKl.

Developmental effects of neonatal isoflurane and sevoflurane exposure in rats.

Background:The general anesthetics, isoflurane and sevoflurane, cause developmental abnormalities in neonatal animal models via incompletely understood mechanisms. Despite many common molecular targets, isoflurane and sevoflurane exhibit substantial differences in their actions. The authors sought to determine whether these differences can also be detected at the level of neurodevelopmental effects. Methods:Postnatal rats, 4–6 days old, were exposed to 1.2% isoflurane or 2.1% sevoflurane for 1–6 h and studied for immediate and delayed effects. Results:Isoflurane exposure was associated with weaker seizure-like electroencephalogram patterns than sevoflurane exposure. Confronted with a new environment at a juvenile age, the sevoflurane-exposed rats spent significantly more time in an “immobile” state than unexposed rats. Electroencephalographic (mean ± SE, 55.5 ± 12.80 s vs. 14.86 ± 7.03 s; P = 0.014; n = 6–7) and spontaneous behavior (F(2,39) = 4.43; P = 0.018) effects of sevoflurane were significantly diminished by pretreatment with the Na+–K+–2Cl– cotransporter inhibitor bumetanide, whereas those of isoflurane were not. Pretreatment with bumetanide, however, diminished isoflurane-induced activation of caspase-3 in the cerebral cortex (F(2,8) = 22.869; P = 0.002) and prevented impairment in sensorimotor gating function (F(2,36) = 5.978; P = 0.006). Conclusions:These findings in combination with results previously reported by the authors suggest that isoflurane and sevoflurane produce developmental effects acting via similar mechanisms that involve an anesthetic-induced increase in neuronal activity. At the same time, differences in their effects suggest differences in the mediating mechanisms and in their relative safety profile for neonatal anesthesia.

L-phenylalanine selectively depresses currents at glutamatergic excitatory synapses

To explore the hypothesis that L‐phenylalanine (L‐Phe) depresses glutamatergic synaptic transmission and thus contributes to brain dysfunction in phenylketonuria (PKU), the effects of L‐Phe on spontaneous and miniature excitatory postsynaptic currents (s/mEPSCs) in rat and mouse hippocampal and cerebrocortical cultured neurons were studied using the patch‐clamp technique. L‐Phe depressed the amplitude and frequency of both N‐methyl‐D‐aspartate (NMDA) and non‐NMDA components of glutamate receptor (GluR) s/mEPSCs. The IC50 of L‐Phe to inhibit non‐NMDAR mEPSC frequency was 0.98 ± 0.13 mM, a brain concentration seen in classical PKU. In contrast, D‐Phe had a significantly smaller effect, whereas L‐leucine, an amino acid that competes with L‐Phe for brain transporter, had no effect on mEPSCs. Unlike GluR s/mEPSCs, GABA receptor mIPSCs were not attenuated by L‐Phe. A high extracellular concentration of glycine prevented the attenuation by L‐Phe of NMDAR current, activated by exogenous agonist, and of NMDAR s/mEPSC amplitude, but not of NMDAR s/mEPSC frequency. On the other hand, L‐Phe significantly depressed non‐NMDAR current activated by low but not high concentrations of exogenous agonists. Glycine‐independent attenuation of NMDAR s/mEPSC frequency suggests decreased presynaptic glutamate release caused by L‐Phe, whereas decreased amplitudes of NMDAR and non‐NMDAR s/mEPSCs are consistent with competition of L‐Phe for the glycine‐ and glutamate‐binding sites of NMDARs and non‐NMDARs, respectively. The finding that GluR activity is significantly depressed at conditions characteristic of classical PKU indicates a potentially important contribution of impaired GluR function to PKU‐related mental retardation and provides important insights into the potential physiological consequences of impaired GluR function.

Roles of aldosterone and oxytocin in abnormalities caused by sevoflurane anesthesia in neonatal rats.

Background:The authors sought to determine whether subjects with pathophysiological conditions that are characterized by increased concentrations of aldosterone have increased susceptibility to the side effects of neonatal anesthesia with sevoflurane. Methods:Postnatal day 4–20 (P4–P20) rats were exposed to sevoflurane, 6% and 2.1%, for 3 min and 60–360 min, respectively. Exogenous aldosterone was administered to imitate pathophysiological conditions with increased concentrations of aldosterone. Results:Six hours of anesthesia with sevoflurane on P4–P5 rats resulted in a more than 30-fold increase in serum concentrations of aldosterone (7.02 ± 1.61 ng/dl vs. 263.75 ± 22.31 ng/dl, mean ± SE, n = 5–6) and reduced prepulse inhibition of the acoustic startle response (F(2,37) = 5.66, P < 0.001). Administration of exogenous aldosterone during anesthesia with sevoflurane enhanced seizure-like electroencephalogram patterns in neonatal rats (48.25 ± 15.91 s vs. 222.00 ± 53.87 s, mean ± SE, n = 4) but did not affect electroencephalographic activity in older rats. Exogenous aldosterone increased activation of caspase-3 (F(3,28) = 11.02, P < 0.001) and disruption of prepulse inhibition of startle (F(3,46) = 6.36; P = 0.001) caused by sevoflurane. Intracerebral administration of oxytocin receptor agonists resulted in depressed seizure-like electroencephalogram patterns (F(2,17) = 6.37, P = 0.009), reduced activation of caspase-3 (t(11) = 2.83, P = 0.016), and disruption of prepulse inhibition of startle (t(7) = −2.9; P = 0.023) caused by sevoflurane. Conclusions:These results suggest that adverse developmental effects of neonatal anesthesia with sevoflurane may involve both central and peripheral actions of the anesthetic. Subjects with increased concentrations of aldosterone may be more vulnerable, whereas intracerebral oxytocin receptor agonists may be neuroprotective.

Epilepsy in Phenylketonuria: A Complex Dependence on Serum Phenylalanine Levels

Summary:  Purpose: Phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism that frequently results in epilepsy if a low Phe diet was not implemented at birth. The mechanisms by which Phe affects the brain are poorly understood.

Specific inhibition of N-methyl-D-aspartate receptor function in rat hippocampal neurons by L-phenylalanine at concentrations observed during phenylketonuria

Hippocampal N-methyl-D-aspartate receptors (NMDARs) are thought to be involved in the regulation of memory formation and learning. Investigation of NMDAR function during experimental conditions known to be associated with impaired cognition in vivo may provide new insights into the role of NMDARs in learning and memory. Specifically, the mechanism whereby high concentrations of L-phenylalanine (L-Phe) during phenylketonuria (>1.2 mM) cause mental retardation remains unknown. Therefore, the effects of L-Phe on NMDA-activated currents (INMDA) were studied in cultured hippocampal neurons from newborn rats using the patch-clamp technique. L-Phe specifically and reversibly attenuated INMDA in a concentration-dependent manner (IC50 = 1.71 ± 0.24 mM). In contrast, L-tyrosine (L-Tyr), an amino acid synthesized from L-Phe in normal subjects, did not significantly change INMDA. Although the L-Phe-INMDA concentration-response relationship was independent of the concentration of NMDA, it was shifted rightward by increasing the concentration of glycine. Consistent with an effect of L-Phe on the NMDAR glycine-binding site, L-Phe (1 mM) did not attenuate INMDA in the presence of D-alanine (10 μM). Furthermore, L-Phe significantly attenuated neither glutamate-activated current in the presence of MK-801, nor current activated by AMPA. The finding that L-Phe inhibits specifically NMDAR current in hippocampal neurons by competing for the glycine-binding site suggests a role for impaired NMDAR function in the development of mental retardation during phenylketonuria and accordingly an important role for NMDARs in memory formation and learning.

PKU is a reversible neurodegenerative process within the nigrostriatum that begins as early as 4 weeks of age in Pahenu2 mice

Phenylketonuria (PKU) is a common genetic disorder in humans that arises from deficient activity of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination. The neuropathogenesis of the disease has not been completely elucidated, however, oxidative stress is considered to be a key feature of the disease process. Hyperphenylalanemia also adversely affects monoaminergic metabolism in the brain. For this reason we chose to evaluate the nigrostriatum of Pah(enu2) mice, to determine if alterations of monoamine metabolism resulted in morphologic nigrostriatal pathology. Furthermore, we believe that recent developments in adeno-associated virus (AAV)-based vectors have greatly increased the potential for long-term gene therapy and may be a viable alternative to dietary treatment for this metabolic disorder. In this study we identified neurodegenerative changes with regenerative responses in the nigrostriatum of Pah(enu2) mice that are consistent with oxidative injury and occurred as early as 4 weeks of age. These neuropathologic changes were reversed following portal vein delivery of a recombinant adeno-associated virus-mouse phenylalanine hydroxylase-woodchuck hepatitis virus post-transcriptional response element (rAAV-mPAH-WPRE) vector to Pah(enu2) mice and corresponded to rapid reduction of serum Phe levels.

Neuroprotective Action of Halogenated Derivatives of L-Phenylalanine

Background and Purpose— The aromatic amino acid L-Phenylalanine (L-Phe) significantly and reversibly depresses excitatory glutamatergic synaptic transmission (GST) via a unique set of presynaptic and postsynaptic mechanisms. Therefore, we hypothesized that endogenous derivatives of L-Phe, which display potent antiglutamatergic activity, may safely and efficaciously protect the brain during conditions characterized by overactivation of glutamate receptors. Methods— We tested this hypothesis in vitro with a combination of patch-clamp and lactate dehydrogenase (LDH) analyses in rat cultured neurons exposed to simulated ischemia, and in vivo using a rat model of experimental stroke caused by transient middle cerebral artery occlusion (MCAO). Results— 3,5-diiodo-l-tyrosine (DIT) and 3,5-dibromo-l-tyrosine (DBrT), endogenous halogenated derivatives of L-Phe, attenuated GST by similar mechanisms as L-Phe, but with greater potency. For example, the IC50s for DIT and DBrT to depress the frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor-mediated mEPSCs were 104.6±14.1 μmol/L and 127.5±13.3 μmol/L, respectively. Depression of GST by DIT and DBrT persisted during energy deprivation. Furthermore, DBrT significantly reduced LDH release in neuronal cultures exposed to oxygen glucose deprivation. In rats subjected to transient MCAO, DBrT decreased the brain infarct volume and neurological deficit score to 52.7±14.1% and 57.1±12.0% of control values, respectively. DBrT neither altered atrioventricular nodal and intraventricular conduction in isolated heart, nor heart rate and blood pressure in vivo. Conclusion— DBrT, an endogenous halogenated derivative of L-Phe, shows promise as a representative of a novel class of neuroprotective agents by exerting significant neuroprotection in both in vitro and in vivo models of brain ischemia.

Endocrine and neurobehavioral abnormalities induced by propofol administered to neonatal rats.

Background:The authors studied whether neonatal propofol anesthesia affects development of the endocrine and neural systems. Methods:Sprague–Dawley rats were anesthetized using intraperitoneal propofol for 5 h on postnatal days (P) 4, 5, or 6. Pups that received either saline or intralipid, but not those in the negative control groups, were also maternally separated for 5 h. Serum levels of corticosterone were measured immediately after anesthesia and in adulthood after prepulse inhibition of acoustic startle testing (≥P80), followed by measurement of hippocampal neuronal activity. Results:Propofol acutely increased corticosterone levels to 146.6 ± 23.5 ng/ml (n = 6) versus 16.4 ± 3.5 ng/ml (n = 6) and 18.4 ± 3.2 ng/ml (n = 6) in saline- and intralipd-treated pups, respectively. In adulthood, the propofol group exhibited exacerbated endocrine responses to stress in a form of increased corticosterone levels (1,171.58 ± 149.17 ng/ml [n = 15] vs. 370.02 ± 36.01 ng/ml [n = 10] in the saline group). The propofol group had increased the frequency of miniature inhibitory postsynaptic currents in CA1 neurons of male and female rats, but reduced prepulse inhibition of startle was detected only in males. The Na+–K+–2Cl− cotransporter inhibitor bumetanide, administered to pups before propofol injection, alleviated long-term endocrine and prepulse inhibition abnormalities. Exogenous corticosterone, administered to naive pups, induced synaptic and endocrine but not prepulse inhibition effects, similar to those of propofol. Conclusion:Propofol-caused acute increases in corticosterone levels and &ggr;-aminobutyric acid type A receptor–mediated excitation at the time of anesthesia may play mechanistic roles in development of exacerbated endocrine responses to stress and neurobehavioral abnormalities.