Behavioural impairments after exposure of neonatal mice to propofol are accompanied by reductions in neuronal activity in cortical circuitry.

BACKGROUND: Both animal and retrospective human studies have linked extended and repeated general anaesthesia during early development with cognitive and behavioural deficits later in life. However, the neuronal circuit mechanisms underlying this anaesthesia-induced behavioural impairment are poorly understood. METHODS: Neonatal mice were administered one or three doses of propofol, a commonly used i.v. general anaesthetic, over Postnatal days 7-11. Control mice received Intralipid® vehicle injections. At 4 months of age, the mice were subjected to a series of behavioural tests, including motor learning. During the process of motor learning, calcium activity of pyramidal neurones and three classes of inhibitory interneurones in the primary motor cortex were examined in vivo using two-photon microscopy. RESULTS: Repeated, but not a single, exposure of neonatal mice to propofol i.p. caused motor learning impairment in adulthood, which was accompanied by a reduction of pyramidal neurone number and activity in the motor cortex. The activity of local inhibitory interneurone networks was also altered: somatostatin-expressing and parvalbumin-expressing interneurones were hypoactive, whereas vasoactive intestinal peptide-expressing interneurones were hyperactive when the mice were performing a motor learning task. Administration of low-dose pentylenetetrazol to attenuate γ-aminobutyric acid A receptor-mediated inhibition or CX546 to potentiate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-subtype glutamate receptor function during emergence from anaesthesia ameliorated neuronal dysfunction in the cortex and prevented long-term behavioural deficits. CONCLUSIONS: Repeated exposure of neonatal mice to propofol anaesthesia during early development causes cortical circuit dysfunction and behavioural impairments in later life. Potentiation of neuronal activity during recovery from anaesthesia reduces these adverse effects of early-life anaesthesia.

Role of mitochondrial complex I and protective effect of CoQ10 supplementation in propofol induced cytotoxicity.

Propofol (2,6-diisopropylphenol) is an anaesthetic widely used for human sedation. Due to its intrinsic antioxidant properties, rapid induction of anaesthesia and fast recovery, it is employed in paediatric anaesthesia and in the intensive care of premature infants. Recent studies have pointed out that exposure to anaesthesia in the early stage of life might be responsible of long-lasting cognitive impairment. The apoptotic neurodegeneration induced by general anaesthetics (GA) involves mitochondrial impairment due to the inhibition of the OXPHOS machinery. In the present work, we aim to identify the main mitochondrial respiratory chain target of propofol toxicity and to evaluate the possible protective effect of CoQ10 supplementation. The propofol effect on the mitochondrial functionality was assayed in isolated mitochondria and in two cell lines (HeLa and T67) by measuring oxygen consumption rate. The protective effect of CoQ10 was assessed by measuring cells viability, NADH-oxidase activity and ATP/ADP ratio in cells treated with propofol. Our results show that propofol reduces cellular oxygen consumption rate acting mainly on mitochondrial Complex I. The kinetic analysis of Complex I inhibition indicates that propofol interferes with the Q module acting as a non-competitive inhibitor with higher affinity for the free form of the enzyme. Cells supplemented with CoQ10 are more resistant to propofol toxicity. Propofol exposure induces cellular damages due to mitochondrial impairment. The site of propofol inhibition on Complex I is the Q module. CoQ10 supplementation protects cells against the loss of energy suggesting its possible therapeutic role to minimizing the detrimental effects of general anaesthesia.

Anesthetic propofol overdose causes vascular hyperpermeability by reducing endothelial glycocalyx and ATP production.

Prolonged treatment with a large dose of propofol may cause diffuse cellular cytotoxicity; however, the detailed underlying mechanism remains unclear, particularly in vascular endothelial cells. Previous studies showed that a propofol overdose induces endothelial injury and vascular barrier dysfunction. Regarding the important role of endothelial glycocalyx on the maintenance of vascular barrier integrity, we therefore hypothesized that a propofol overdose-induced endothelial barrier dysfunction is caused by impaired endothelial glycocalyx. In vivo, we intraperitoneally injected ICR mice with overdosed propofol, and the results showed that a propofol overdose significantly induced systemic vascular hyperpermeability and reduced the expression of endothelial glycocalyx, syndecan-1, syndecan-4, perlecan mRNA and heparan sulfate (HS) in the vessels of multiple organs. In vitro, a propofol overdose reduced the expression of syndecan-1, syndecan-4, perlecan, glypican-1 mRNA and HS and induced significant decreases in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio and ATP concentrations in human microvascular endothelial cells (HMEC-1). Oligomycin treatment also induced significant decreases in the NAD+/NADH ratio, in ATP concentrations and in syndecan-4, perlecan and glypican-1 mRNA expression in HMEC-1 cells. These results demonstrate that a propofol overdose induces a partially ATP-dependent reduction of endothelial glycocalyx expression and consequently leads to vascular hyperpermeability due to the loss of endothelial barrier functions.

Propofol (Diprivan®) and Intralipid® exacerbate insulin resistance in type-2 diabetic hearts by impairing GLUT4 trafficking.

BACKGROUND: The IV anesthetic, propofol, when administered as fat emulsion-based formulation (Diprivan) promotes insulin resistance, but the direct effects of propofol and its solvent, Intralipid, on cardiac insulin resistance are unknown. METHODS: Hearts of healthy and type-2 diabetic rats (generated by fructose feeding) were aerobically perfused for 60 minutes with 10 µM propofol in the formulation of Diprivan or an equivalent concentration of its solvent Intralipid (25 µM) ± insulin (100 mU•L). Glucose uptake, glycolysis, and glycogen metabolism were measured using [H]glucose. Activation of Akt, GSK3ß, AMPK, ERK1/2, p38MAPK, S6K1, JNK, protein kinase Cθ (PKCθ), and protein kinase CCßII (PKCßII) was determined using immunoblotting. GLUT4 trafficking and phosphorylations of insulin receptor substrate-1 (IRS-1) at Ser307(h312), Ser1100(h1101), and Tyr608(hTyr612) were measured. Mass spectrometry was used to determine acylcarnitines, phospholipids, and sphingolipids. RESULTS: Diprivan and Intralipid reduced insulin-induced glucose uptake and redirected glucose to glycogen stores in diabetic hearts. Reduced glucose uptake was accompanied by lower GLUT4 trafficking to the sarcolemma. Diprivan and Intralipid inactivated GSK3ß but activated AMPK and ERK1/2 in diabetic hearts. Only Diprivan increased phosphorylation of Akt(Ser473/Thr308) and translocated PKCθ and PKCßII to the sarcolemma in healthy hearts, whereas it activated S6K1 and p38MAPK and translocated PKCßII in diabetic hearts. Furthermore, only Diprivan phosphorylated IRS-1 at Ser1100(h1101) in healthy and diabetic hearts. JNK expression, phosphorylation of Ser307(h312) of IRS-1, and PKCθ expression and translocation were increased, whereas GLUT4 expression was reduced in insulin-treated diabetic hearts. Phosphatidylglycerol, phosphatidylethanolamine, and C18-sphingolipids accumulated in Diprivan-perfused and Intralipid-perfused diabetic hearts. CONCLUSIONS: Propofol and Intralipid promote insulin resistance predominantly in type-2 diabetic hearts.

Alphaxalone Reformulated: A Water-Soluble Intravenous Anesthetic Preparation in Sulfobutyl-Ether-ß-Cyclodextrin.

BACKGROUND: Alphaxalone is a neuroactive steroid anesthetic that is poorly water soluble. It was formulated in 1972 as Althesin® using Cremophor® EL, a nonionic surfactant additive. The product was a versatile short-acting IV anesthetic used in clinical practice in many countries from 1972 to 1984. It was withdrawn from clinical practice because of hypersensitivity to Cremophor EL. In the investigations reported here, we compared the properties of 3 anesthetics: a new aqueous solution of alphaxalone dissolved in 7-sulfobutyl-ether-ß-cyclodextrin (SBECD, a water-soluble molecule with a lipophilic cavity that enables drug solubilization in water); a Cremophor EL preparation of alphaxalone; and propofol. METHODS: Two solutions of alphaxalone (10 mg/mL) were prepared: one using 13% w/v solution of SBECD in 0.9% saline (PHAX) and the other a solution of alphaxalone prepared as described in the literature using 20% Cremophor EL (ALTH). A solution of propofol (10 mg/mL; PROP) in 10% v/v soya bean oil emulsion was used as a comparator anesthetic. Jugular IV catheters were implanted in male Wistar rats (180-220 g) under halothane anesthesia. Separate groups of 10 implanted rats each were given IV injections of PHAX, ALTH, or PROP from 1.2 mg/kg to lethal doses. Doses of each drug that caused anesthesia (loss of righting reflex and response to tail pinch) and lethality in 50% of rats were calculated by probit analysis. The drugs were also compared for effects on arterial blood pressure and heart rate. RESULTS: IV PHAX, ALTH, and PROP caused dose-related sedation and anesthesia, with 50% effective dose (ED50) values for loss of righting reflex being 2.8, 3.0, and 4.6 mg/kg, respectively. PROP led to death in 10 of 10 rats at doses >30 mg/kg (50% lethal dose (LD50) = 27.7 mg/kg). A dose of alphaxalone 53 mg/kg as ALTH caused 10 of 10 rats to die (LD50 = 43.6 mg/kg), whereas none died when given the same doses of alphaxalone formulated in SBECD. PHAX caused 20% lethality at the maximal dose tested of 84 mg/kg. PHAX caused less cardiovascular depression than PROP. Control experiments with the 3 drug-free vehicles showed no effects. CONCLUSIONS: Alphaxalone caused fast-onset anesthesia at the same dose for both formulations (PHAX and ALTH). The use of SBECD as a drug-solubilizing excipient did not alter the anesthetic effect of alphaxalone, but it did increase the therapeutic index of alphaxalone in PHAX compared with ALTH. PHAX has a higher safety margin than the propofol lipid formulation and also the alphaxalone formulation in Cremophor EL (ALTH).

Protective effect of acetyl-L-carnitine on propofol-induced toxicity in embryonic neural stem cells.

Propofol is a widely used general anesthetic. A growing body of data suggests that perinatal exposure to general anesthetics can result in long-term deleterious effects on brain function. In the developing brain there is evidence that general anesthetics can cause cell death, synaptic remodeling, and altered brain cell morphology. Acetyl-L-carnitine (L-Ca), an anti-oxidant dietary supplement, has been reported to prevent neuronal damage from a variety of causes. To evaluate the ability of L-Ca to protect against propofol-induced neuronal toxicity, neural stem cells were isolated from gestational day 14 rat fetuses and on the eighth day in culture were exposed for 24h to propofol at 10, 50, 100, 300 and 600 µM, with or without L-Ca (10 µM). Markers of cellular proliferation, mitochondrial health, cell death/damage and oxidative damage were monitored to determine: (1) the effects of propofol on neural stem cell proliferation; (2) the nature of propofol-induced neurotoxicity; (3) the degree of protection afforded by L-Ca; and (4) to provide information regarding possible mechanisms underlying protection. After propofol exposure at a clinically relevant concentration (50 µM), the number of dividing cells was significantly decreased, oxidative DNA damage was increased and a significant dose-dependent reduction in mitochondrial function/health was observed. No significant effect on lactase dehydrogenase (LDH) release was observed at propofol concentrations up to 100 µM. The oxidative damage at 50 µM propofol was blocked by L-Ca. Thus, clinically relevant concentrations of propofol induce dose-dependent adverse effects on rat embryonic neural stem cells by slowing or stopping cell division/proliferation and causing cellular damage. Elevated levels of 8-oxoguanine suggest enhanced oxidative damage [reactive oxygen species (ROS) generation] and L-Ca effectively blocks at least some of the toxicity of propofol, presumably by scavenging oxidative species and/or reducing their production.

Additives in intravenous anesthesia modulate pulmonary inflammation in a model of LPS-induced respiratory distress.

BACKGROUND: It has been suggested that propofol with ethylenediaminetetraacetic acid (EDTA) can modulate the systemic inflammatory response. Prolonged higher levels of pulmonary inflammation are associated with poor outcome of patients with acute lung injury. In the present study, we hypothesized that pulmonary inflammation could be modulated by propofol with EDTA compared with propofol with sulfite. METHODS: Respiratory distress was induced in rats (n=25) by intratracheal nebulization of lipopolysaccharide (LPS). After 24 h, animals were randomized to either propofol with EDTA (Propofol(EDTA)), propofol with sulfite (Propofol(sulfite)) or ketamine/midazolam (Ket/Mid); control animals received saline (n=30). Animals were ventilated for 4 h and blood gases were measured hourly. Bronchoalveolar lavage (BAL) was performed for cytokine analysis of: tumor necrosis factor (TNF), interleukin (IL)-6 and macrophage inflammatory protein (MIP)-2. RESULTS: LPS led to increased pulmonary inflammation in all groups compared with the control groups. Gas exchange deteriorated over time only in the LPS Propofol(sulfite) group and was significantly lower than the Ket/Mid group. Only IL-6 was significantly higher in the LPS Propofol(sulfite) group compared with both the Ket/Mid group and the Propofol(EDTA) group. CONCLUSION: Pulmonary IL-6 can be modulated by additives in systemic anesthesia. IMPLICATION STATEMENT: This study demonstrates that pulmonary inflammation caused by direct lung injury can be modulated by intravenous anesthesia used in critically ill patients.