Morten Larsen Vinje

Volatile anaesthetics depolarize neural mitochondria by inhibiton of the electron transport chain

Background:  The mitochondrial membrane potential (ΔΨm) controls the generation of adenosine triphosphate (ATP) and reactive oxygen species, and sequesteration of intracellular Ca2+[Ca2+]i. Clinical concentrations of sevoflurane affect the ΔΨm in neural mitochondria, but the mechanisms remain elusive. The aim of the present study was to compare the effect of isoflurane and sevoflurane on ΔΨm in rat pre‐synaptic terminals (synaptosomes), and to investigate whether these agents affect ΔΨm by inhibiting the respiratory chain.

Depolarization of mitochondria in isolated CA1 neurons during hypoxia, glucose deprivation and glutamate excitotoxicity.

During cerebral ischemia neuronal injury is induced by a combination of hypoxia, hypoglycemia and glutamate excitotoxicity. To evaluate the relative importance of these factors on the mitochondrial function, acutely isolated rat hippocampal CA1 neurons were loaded with Rhodamine 123 to monitor the mitochondrial membrane potential (Deltapsim). During 15 min of hypoxia, a rapid and complete mitochondrial depolarization was observed in all neurons also when complex V of the respiratory chain was blocked by oligomycin. Glucose deprivation caused 77% of the neurons to loose the Deltapsim completely, whereas most oligomycin-treated neurons retained their Deltapsim. During oxygen and glucose deprivation, a similar mitochondrial response was seen as in hypoxia. Although 15 min of high glutamate concentration (1 mM) provoked a rapid and irreversible increase in [Ca2+]i, only 25% of the neurons lost the Deltapsim. All oligomycin-treated neurons, however, lost the Deltapsim during glutamate exposure. In conclusion, the mitochondrial function of acutely isolated CA1 neurons is more sensitive to hypoxia than to glucose deprivation and glutamate excitotoxicity.

The effect of sevoflurane on glutamate release and uptake in rat cerebrocortical presynaptic terminals

Background: Volatile anaesthetics exert their effect in the brain mainly by reducing synaptic excitability. Isoflurane abates excitation by reducing the release and increasing the uptake of transmitter glutamate into the presynaptic terminal. The exact molecular mechanisms exerting these effects, however, are not clear. Voltage‐gated calcium channels have been proposed as the pharmacological target. The present study examines the effect of sevoflurane on synaptic glutamate release and free cytosolic calcium and the effect on high‐ and low‐affinity uptake of L‐glutamate using isolated presynaptic terminals prepared from rat cerebral cortex.

Sevoflurane and propofol depolarize mitochondria in rat and human cerebrocortical synaptosomes by different mechanisms.

Background and objectives: The mitochondrial membrane potential drives the main functions of the mitochondria. Sevoflurane depolarizes neural mitochondria. There is still, however, limited information concerning the effect of anaesthetics on neural mitochondria in humans. The effect of sevoflurane and propofol on the intracellular Ca2+ concentration [Ca2+]i and the mitochondrial membrane potential (ΔΨm) was therefore compared in rat and human synaptosomes, and the changes were related to interventions in the electron transport chain.

Endoplasmic Reticulum Dysfunction and Ca2+ Deregulation in Isolated CA1 Neurons during Oxygen and Glucose Deprivation

Intracellular calcium ([Ca2+]i) plays a pivotal role in neuronal ischemia. The aim of the present study was to investigate the routes of Ca2+ entry during non-excitotoxic oxygen and glucose deprivation (OGD) in acutely dissociated rat CA1 neurons. During OGD the fluo-3/fura red ratio reflecting [Ca2+]i increased rapidly and irreversibly. [Ca2+]i increased to the same degree in Ca2+ depleted medium, and also when both the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate (IP3) receptors were blocked. When the endoplasmic reticulum (ER) Ca2+ stores were emptied with thapsigargin no increase in [Ca2+]i was observed independent of extracellular Ca2+. The OGD induced Ca2+ deregulation in isolated CA1 neurons is not prevented by removing Ca2+, or by blocking the IP3– or RyR receptors. However, when SERCA was blocked, no increase in [Ca2+]i was observed suggesting that SERCA dysfunction represents an important mechanism for ischemic Ca2+ overload.

Calcium-dependent protein kinase C activation in acutely isolated neurons during oxygen and glucose deprivation.

Glutamate excitotoxicity and necrotic cell death are characteristic features of ischemic neuronal injury. In the penumbral area, glutamate exposure is less pronounced and neuronal death is delayed. Recent studies suggest that delayed neuronal death is propagated by intracellular signalling pathways. Protein kinase C (PKC) activation may initiate apoptosis, but its role in ischemia is still not clear. In this study the PKC activity was investigated during non-excitotoxic ischemia in acutely dissociated rat CA1 neurons. During oxygen and glucose deprivation (OGD.05), but with a slower onset. In neurons treated with thapsigargin in a Ca2+ depleted solution, however, OGD did not trigger PKC activation. The results suggest that the PKC activation is mainly triggered by Ca2+ release from endogenous stores.

Sevoflurane reduces synaptic glutamate release in human synaptosomes.

Volatile anesthetics reduce excitatory synaptic transmission in the mammalian brain. In the present study, the effect of sevoflurane on synaptic glutamate release, free cytosolic Ca2+ ([Ca2+]i), and glutamate uptake was investigated using isolated presynaptic terminals prepared from human cerebral cortex. The tissue was obtained from standard temporal lobe specimens removed because of epilepsy. The glutamate release and [Ca2+]i was measured as the fluorescence of nicotinamide adenine dinucleotide phosphate (NADPH) and fura-2, respectively. The uptake of radiolabeled glutamate was measured in a &bgr;-scintillation counter. Membrane depolarization with 4-aminopyridine for three minutes evoked a Ca2+-dependent glutamate release of 3.4 ± 0.5 nmol/mg. Sevoflurane 2.5 and 4.0% attenuated the evoked release by 45 and 55%, respectively. The evoked increase in [Ca2+]i was not significantly altered by the anesthetic agent. The uptake studies were performed in the high-affinity area, and Km was calculated to 19.3 ± 5.7 × 10−6 M and Vmax to 5.7 ± 1.0 &mgr;mol g−1 min−1. The Km and Vmax values were not significantly altered by sevoflurane 2.5%. These results demonstrate that sevoflurane in the human brain reduces Ca2+-dependent glutamate release. The exact mode of action is still to be resolved.

The effect of isoflurane and sevoflurane on cerebrocortical presynaptic Ca2+ and protein kinase C activity.

&NA; Protein kinase C (PKC) is an important enzyme involved in the regulation of neurotransmission and might also be important in the mediation of ischemic neuronal death. PKC has been implicated as a target of volatile anesthetics as well as in anesthetic protection against ischemia. The present study tested the effect of isoflurane and sevoflurane, both used in neuroanesthetic practice, on presynaptic free cytosolic Ca2+ ([Ca2+]i) and PKC activity. To measure [Ca2+]i and PKC activation simultaneously, rat synaptosomes, mostly containing presynaptic terminals, were loaded with the fluorescent probes fura‐2 and fim‐1, respectively. The synaptosomes were exposed to either isoflurane or sevoflurane in concentrations corresponding to 1 and 2 MAC values in rats, both in Ca2+‐containing and Ca2+‐free medium. After 8 minutes of anesthetic exposure, 1 mM 4‐aminopyridine was added to induce membrane depolarization. Isoflurane 1 and 2 MAC increased the basal PKC activity after 8 minutes in Ca2+‐containing medium by 15.1% (3.6%) and 30.5% (5.5%) compared with control, respectively [mean (SEM); n = 9, both values P < 0.05]. Sevoflurane 2 MAC transiently decreased but thereafter increased the PKC activity (P < 0.05). In Ca2+ ‐free medium sevoflurane attenuated the PKC activity (P < 0.05). The anesthetics did not alter the depolarization‐evoked enzyme activation. Furthermore, 2 MAC of both isoflurane and sevoflurane increased the basaland attenuated the depolarization‐evoked increase in [Ca2+]i (P < 0.05). The present study supports the hypotheses that volatile anesthetics affect presynaptic PKC activity and that the anesthetic effect on enzyme activation seems to be related to an increase in [Ca2+]i.

Measured increase in intracellular Ca2+ during stimulated release of endogenous glutamate from human cerebrocortical synaptosomes

Presynaptic terminals (synaptosomes) prepared from guinea pig and rat cerebral cortex release endogenous glutamate in a Ca(2+)-dependent manner in response to membrane depolarisation. In the present study, synaptosomes were prepared from human cerebral cortex removed in association with temporal lobe resections in epileptic patients. The cytosolic free Ca(2+) concentration increased from 474+/-66 before to 649+/-89 nM after 2 min depolarisation. The basal level of free cytosolic Ca(2+) is higher and the increase in response to depolarisation is more pronounced in human synaptosomes than observed in animal experiments. The Ca(2+)-dependent glutamate release, estimated as the difference between total - and the Ca(2+)-independent glutamate release, increased from 0 to 5.4+/-1.9 nmol/mg protein. The released amount of glutamate is larger than reported in animal models. These results demonstrate that membrane depolarisation of synaptosomes from human brain evokes a rapid rise in cytosolic free Ca(2+) and a more prolonged rise in synaptic, Ca(2+)-dependent glutamate release.

Sevoflurane depolarizes pre‐synaptic mitochondria in the central nervous system

Background:  Volatile anaesthetics protect the heart from ischaemic injury by activating mitochondrial signalling pathways. The aim of this study was to test whether sevoflurane, which is increasingly used in neuroanaesthesia, affects mitochondrial function in the central nervous system by altering the mitochondrial membrane potential (ΔΨm).