Cre-dependent ACR2-expressing reporter mouse strain for efficient long-lasting inhibition of neuronal activity.

Optogenetics is a powerful tool for manipulating neuronal activity by light illumination with high temporal and spatial resolution. Anion-channelrhodopsins (ACRs) are light-gated anion channels that allow researchers to efficiently inhibit neuronal activity. A blue light-sensitive ACR2 has recently been used in several in vivo studies; however, the reporter mouse strain expressing ACR2 has not yet been reported. Here, we generated a new reporter mouse strain, LSL-ACR2, in which ACR2 is expressed under the control of Cre recombinase. We crossed this strain with a noradrenergic neuron-specific driver mouse (NAT-Cre) to generate NAT-ACR2 mice. We confirmed Cre-dependent expression and function of ACR2 in the targeted neurons by immunohistochemistry and electrophysiological recordings in vitro, and confirmed physiological function using an in vivo behavioral experiment. Our results show that the LSL-ACR2 mouse strain can be applied for optogenetic inhibition of targeted neurons, particularly for long-lasting continuous inhibition, upon crossing with Cre-driver mouse strains. The LSL-ACR2 strain can be used to prepare transgenic mice with homogenous expression of ACR2 in targeted neurons with a high penetration ratio, good reproducibility, and no tissue invasion.

General anesthesia with remimazolam for a pediatric patient with MELAS and recurrent epilepsy: a case report.

BACKGROUND: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disease. We report here the safe use of remimazolam in a pediatric MELAS patient. CASE PRESENTATION: A 10-year-old girl (118 cm, 16 kg) was scheduled for an open gastrostomy to improve nutrition and epileptic seizure control. We induced and maintained general anesthesia with remimazolam, remifentanil, fentanyl, and rocuronium. We also performed a bilateral subcostal transversus abdominis plane block before the surgery. The surgery finished uneventfully. After we discontinued remimazolam administration, the patient woke up immediately but calmly without flumazenil. Epileptic seizures did not occur during intra- and early post-operative periods. CONCLUSION: Remimazolam enabled us to provide a pediatric MELAS patient with general anesthesia without causing delayed emergence or epileptic seizures.

Isoflurane activates sarcolemmal adenosine triphosphate-sensitive potassium channels in vascular smooth muscle cells: a role for protein kinase A.

BACKGROUND: Recent evidence indicates that vascular adenosine triphosphate-sensitive potassium (K(ATP)) channels in vascular smooth muscle cells are critical in the regulation of vascular tonus under both physiologic and pathophysiologic conditions. Studies of the interaction of volatile anesthetics with vascular K(ATP) channels have been limited. In the current study, the authors investigated the molecular mechanism of isoflurane's action on vascular K(ATP) channels. METHODS: Electrophysiologic experiments were performed using cell-attached and inside-out patch clamp techniques to monitor native vascular K(ATP) channels, and recombinant K(ATP) channels comprised of inwardly rectifying potassium channel subunits (Kir6.1) and the sulfonylurea receptor (SUR2B). Isometric tension experiments were performed in rat thoracic aortic rings without endothelium. RESULTS: Application of isoflurane (0.5 mM) to the bath solution during cell-attached recordings induced a significant increase in K(ATP) channel activity, which was greatly reduced by pretreatment with a selective inhibitor of protein kinase A (PKA), Rp-cAMPS (100 microM). In inside-out patches, isoflurane did not activate K(ATP) channels. Isoflurane significantly activated wild-type recombinant SUR2B/Kir6.1 in cell-attached patches. Isoflurane-induced activation of wild-type channels was diminished in the PKA-insensitive mutant SUR2B-T633A/Kir6.1, SUR2B-S1465A/Kir6.1, and SUR2B/Kir6.1-S385A. In addition, the authors demonstrated that isoflurane-induced PKA activation was associated with isoflurane-induced decreases in isometric tension in the rat aorta. CONCLUSION: These results indicate that isoflurane activates K(ATP) channels via PKA activation. PKA-dependent vasodilation induced by isoflurane also was observed in isometric tension experiments. Analysis of expressed vascular-type K(ATP) channels suggested that PKA-mediated phosphorylation of both Kir6.1 and SUR2B subunits plays a pivotal role in isoflurane-induced vascular K(ATP) channel activation.

Differential effects of etomidate and midazolam on vascular adenosine triphosphate-sensitive potassium channels: isometric tension and patch clamp studies.

BACKGROUND: The aim of this study was to investigate the effects of two imidazoline-derived intravenous anesthetics, etomidate and midazolam, on vascular adenosine triphosphate-sensitive potassium (KATP) channel activity. METHODS: In isolated rat aorta, isometric tension was recorded to examine the anesthetic effects on vasodilator response to levcromakalim, a selective KATP channel opener. Using the patch clamp method, the anesthetic effects were also examined on the currents through (1) native vascular KATP channels, (2) recombinant KATP channels with different combinations of various types of inwardly rectifying potassium channel (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor (SUR1, 2A, 2B) subunits, (3) SUR-deficient channels derived from a truncated isoform of Kir6.2 subunit (Kir6.2DeltaC36 channels), and (4) mutant Kir6.2DeltaC36 channels with reduced sensitivity to adenosine triphosphate (Kir6.2DeltaC36-K185Q channels). RESULTS: Etomidate (> or = 10 m), but not midazolam (up to 10 m), inhibited the levcromakalim-induced vasodilation, which was sensitive to glibenclamide (IC50: 7.21 x 10 m; maximum inhibitory concentration: 1.22 x 10 m). Etomidate (> or = 3 x 10 m), but not midazolam (up to 10 m), inhibited the native KATP channel activity in both cell-attached and inside-out configurations with IC50 values of 1.68 x 10 m and 1.52 x 10 m, respectively. Etomidate (10 m) also inhibited the activity of various types of recombinant SUR/Kir6.0KATP channels, Kir6.2DeltaC36 channels, and Kir6.2DeltaC36-K185Q channels with equivalent potency. CONCLUSIONS: Clinical concentrations of etomidate, but not midazolam, inhibit the KATP channel activity in vascular smooth muscle cells. The inhibition is presumably through its effects on the Kir6.0 subunit, but not on the SUR subunit, with the binding site different from adenosine triphosphate at the amino acid level.

[Bronchospasm during anesthesia in a patient with Pena-Shokeir syndrome].

A 3-month-old boy with Pena-Shokeir syndrome underwent tracheotomy under general anesthesia. Patients with this syndrome may present anesthetic problems involving difficulties in tracheal intubation, possibilities of malignant hyperthermia, as well as perioperative respiratory complications related to hypoplasia of the lung. General anesthesia was induced and maintained with sevoflurane (2-3%) and nitrous oxide (0-50%) in oxygen (50-100%). The patient developed bronchospasm during tracheotomy. Atropine and epinephrine were administered intravenously and 5% sevoflurane was inhaled. The bronchospasm was improved gradually and surgery was successfully finished. Pena-Shokeir syndrome is an uncommon disease first reported by Pena & Shokeir in 1974 and characterized by congenital multiple arthrogryposis, characteristic facies, camptodactyly and pulmonary hypoplasia. In the perioperative management for a patient with Pena-Shokeir syndrome, special attention should be paid to abnormalities in the upper and lower respiratory systems, especially bronchospasm.

[Relationship between driving pressure and tidal volume during high frequency jet ventilation for the tracheal resection].

BACKGROUND: High frequency jet ventilation (HFJV) is a method of ventilation for tracheal resection because it provides a good surgical field and decreases surgical complications. We should know ventilatory settings of HFJV to perform safe respiratory management. In this study we evaluated the relationship between tidal volume (VT) and driving pressure (DP) during HFJV for tracheal resection. METHODS: Twenty patients undergoing tracheal resection under total intravenous anesthesia were studied. Jet pulse was delivered through a 12 or 15 Fr feeding catheter connected to a jet ventilator. We set DP optionally and inspired oxygen concentration 100%, ventilatory frequency 100 x min(-1), and I/T ratio 0.3 or 0.5. In fourteen patients receiving HFJV with 15 Fr catheter we calculated VT by measured PaCO2, reported VCO2 value, and physiological dead space (1.1 x body weight) for each patient and evaluated relationship between VT and DP. RESULTS: HFJV was feasible in all patients. Pneumothrax occurred in one patient. Respiratory complications including pneumonia and respiratory failure were not observed after surgery in all patients. The calculated VT values showed a significant correlation with DP (VT = 48.1 DP + 44.7, r=0.73, P<0.01). CONCLUSIONS: In respiratory management for tracheal resection with HFJV, we can predict DP from VT calculated by expected PaCO2, reported VCO2 value, and physiological dead space. It enables us to perform safe respiratory management.