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In March 2019, SmartTots, a public-private partnership between the US Food and Drug Administration and the International Anesthesia Research Society, hosted a meeting attended by research experts, anaesthesia journal editors, and government agency representatives to discuss the continued need for rigorous preclinical research and the importance of establishing reporting standards for the field of anaesthetic perinatal neurotoxicity. This group affirmed the importance of preclinical research in the field, and welcomed novel and mechanistic approaches to answer some of the field's largest questions. The attendees concluded that summarising the benefits and disadvantages of specific model systems, and providing guidance for reporting results, would be helpful for designing new experiments and interpreting results across laboratories. This expert opinion report is a summary of these discussions, and includes a focused review of current animal models and reporting standards for the field of perinatal anaesthetic neurotoxicity. This will serve as a practical guide and road map for novel and rigorous experimental work.

The sulfonylurea receptor-2 (SUR2) is a subunit of ATP-sensitive potassium channels (KATP) in heart. Mice with the SUR2 gene disrupted (SUR2m) are constitutively protected from ischemia-reperfusion (I/R) cardiac injury. This was surprising because [K.sub.ATP], either sarcolemmal or mitochondrial or both, are thought to be important for cardioprotection. We hypothesized that SUR2m mice have an altered mitochondrial phenotype that protects against I/R. Mitochondrial membrane potential ([DELTA][[PSI].sub.m]), tolerance to [Ca.sup.2+] load, and reactive oxygen species (ROS) generation were studied by fluorescence-based assays, and volumetric changes in response to [K.sup.+] were measured by light scattering in isolated mitochondria. For resting SUR2m mitochondria compared with wild type, the [DELTA][[PSI].sub.m] was less polarized (46.1 [+ or -] 0.4 vs. 51.9 [+ or -] 0.6%), tolerance to [Ca.sup.2+] loading was increased (163 [+ or -] 2 vs. 116 [+ or -] 2 [micro]M), and ROS generation was enhanced with complex I [8.5 [+ or -] 1.2 vs. 4.9 [+ or -] 0.2 arbitrary fluorescence units (afu)/s] or complex II (351 [+ or -] 51.3 vs. 166 [+ or -] 36.2 afu/s) substrates. SUR2m mitochondria had greater swelling in [K.sup.+] medium (30.2 [+ or -] 3.1%) compared with wild type (14.5 [+ or -] 0.6%), indicating greater [K.sup.+] influx. Additionally, [DELTA][[PSI].sub.m] decreased and swelling increased in the absence of ATP in SUR2m, but the sensitivity to ATP was less compared with wild type. When the mitochondria were subjected to hypoxia-reoxygenation, the decrease in respiration rates and respiratory control index was less in SUR2m. [DELTA][[PSI].sub.m] maintenance in the SUR2m intact myocytes was also more tolerant to metabolic inhibition. In conclusion, the cardioprotection observed in the SUR2m mice is associated with a protected mitochondrial phenotype resulting from enhanced K+ conductance that partially dissipated [DELTA][[PSI].sub.m]. These results have implications for possible SUR2 participation in mitochondrial [K.sub.ATP]. adenosine 5-triphosphate-sensitive potassium channel; sulfonylurea receptor 2; ischemia; ischemic-preconditioning; mitochondria; mitochondrial membrane potential; mitochondrial respiration; cellular energetics doi: 10.1152/ajpheart.00363.2010.

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial [Ca.sup.2+] overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential ([DELTA][[PSI].sub.m]) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS. [Ca.sup.2+], and mPTP opening. The magnitude of APC-induced decrease in [DELTA][[PSI].sub.m] was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in [DELTA][[PSI].sub.m]. In cardiomyocytes, [DELTA][[PSI].sub.m], ROS, mPTP opening, and cytosolic and mitochondrial [Ca.sup.2+] were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and [Ca.sup.2+] uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not [Ca.sup.2+] uptake. However, in stressed cardiomyocytes, a similar decrease in [DELTA][[PSI].sub.m] attenuated both cytosolic and mitochondrial [Ca.sup.2+] accumulation. In conclusion, a partial decrease in [DELTA][[PSI].sub.m] underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in [DELTA][[PSI].sub.m] primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial [Ca.sup.2+] uptake. cardioprotection; oxidative stress; mitochondria; reactive oxygen species doi: 10.1152/ajpcell.00006.2010.

From time of their discovery, sarcolemmal ATP-sensitive [K.sup.+] ([sarcK.sub.ATP]) channels were thought to have an important protective role in the heart during stress whereby channel opening protects the heart from stress-induced [Ca.sup.2+] overload and resulting damage. In contrast, some recent studies indicate that [sarcK.sub.ATP] channel closing can lead to cardiac protection. Also, the role of the [sarcK.sub.ATP] channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the [sarcK.sub.ATP] channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of [sarcK.sub.ATP] channel inhibitor HMR-1098 as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol, suggesting activation of the mitochondrial death pathway, [sarcK.sub.ATP] channel inhibition during stress had no effect on Bcl-2, Bad, and phospho-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of the apoptotic pathway. However, monitoring of mitochondrial [Ca.sup.2+] with rhod-2 fluorescent indicator revealed that mitochondrial [Ca.sup.2+] accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of [sarCK.sub.ATP] channels, through a specific [Ca.sup.2+]-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress. cardioprotection; mitochondrial calcium; cardiomyocytes

Ischemic cardiac injury can be substantially alleviated by exposing the heart to pharmacological agents such as volatile anesthetics before occurrence of ischemia-reperfusion. A hallmark of this preconditioning phenomenon is its memory, when cardioprotective effects persist even after removal of preconditioning stimulus. Since numerous studies pinpoint mitochondria as crucial players in protective pathways of preconditioning, the aim of this study was to investigate the effects of preconditioning agent isoflurane on the mitochondrial bioenergetic phenotype. Endogenous flavoprotein fluorescence, an indicator of mitochondrial redox state, was elevated to 195 [+ or -] 16% of baseline upon isoflurane application in intact cardiomyocytes, indicating more oxidized state of mitochondria. Isoflurane treatment also elicited partial dissipation of mitochondrial transmembrane potential, which remained depolarized even after anesthetic withdrawal (tetramethyl-rhodamine fluorescence intensity declined to 83 [+ or -] 3 and 81 [+ or -] 7% of baseline during isoflurane exposure and washout, respectively). Mild uncoupling, with preserved ATP synthesis, was also detected in mitochondria that were isolated from animals that had been previously preconditioned by isoflurane in vivo, revealing its memory nature. These mitochondria, after exposure to hypoxia and reoxygenation, exhibited better preserved respiration and ATP synthesis compared with mitochondria from nonpreconditioned animals. Partial mitochondrial depolarization was paralleled by a diminished [Ca.sup.2+] uptake into isoflurane-treated mitochondria, as indicated by the reduced increment in rhod-2 fluorescence when mitochondria were challenged with increased [Ca.sup.2+] (180 [+ or -] 24 vs. 258 [+ or -] 14% for the control). In conclusion, isoflurane preconditioning elicits partial mitochondrial uncoupling and reduces mitochondrial [Ca.sup.2+] uptake. These effects are likely to reduce the extent of the mitochondrial damage after the hypoxic stress. cardioprotection; uncoupling doi: 10.1152/ajpcell.00221.2006.

Activation of the mitochondrial ATP-sensitive [K.sup.+] channel (mito[K.sub.ATP],) and its regulation by PKC are critical events in preconditioning induced by ischemia or pharmaceutical agents in animals and humans. The properties of the human cardiac mito[K.sub.ATP] channel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mito[K.sub.ATP] channel located in the inner mitochondrial membrane (IMM) due to the physical barrier of the outer mitochondrial membrane. In the present study, we characterized the human cardiac mito[K.sub.ATP] channel and its potential regulation by PKC associated with the IMM. IMM fractions isolated from human left ventricles were fused into lipid bilayers in symmetrical potassium glutamate (150 mM). The conductance of native mito[K.sub.ATP] channels was usually below 80 pS (~70%), which was reduced by ATP and 5-hydroxydecanoic acid (5-HD) in a dose- and time-dependent manner. The native mito[K.sub.ATP] channel is activated by diazoxide and inhibited by ATP and 5-HD. The PKC activator phorbol 12-myristate 13-acetate (2 [micro]M) increased the cumulative open probability of the mito[K.sub.ATP] channel previously inhibited by ATP (P < 0.05), but its inactive analog 4[alpha]-phorbol 12,13-didecanoate had no effect. Western blot analysis detected an inward rectifying [K.sup.+] channel (Kir6.2) immunoreactive protein at 56 kDa and PKC-[delta] in the IMM. These data provide the first characterization of the human cardiac mito[K.sub.ATP] channel and its regulation by PKC(s) in IMM. This local PKC control mechanism may represent an alternative pathway to that proposed previously for cytosolic PKC during ischemic/ pharmacological preconditioning. bilayers; mitochondria; protein kinase C; ischemia; preconditioning

Activation of mitochondrial ATP-sensitive [K.sup.+] (mitO[K.sub.ATP]) channels is critical in myocardial protection induced by preconditioning with volatile anesthetics or brief periods of ischemia. In this study, we characterized rat mito[K.sub.ATP] channels reconstituted in lipid bilayers and examined their direct regulation by isoflurane. Mitochondria and the inner membrane fraction were isolated from rat ventricles and fused into lipid bilayers. On the basis of their inhibition by 5-hydroxydecanoate (5-HD)/ATP or activation by diazoxide, mitO[K.sub.ATP] channels of several conductance states were observed in symmetrical (150 mM) potassium glutamate (26, 47, 66, 83, and 105 pS). Isoflurane (0.8 mM) increased the cumulative open probability from 0.09 [+ or -] 0.02 at baseline to 0.50 [+ or -] 0.09 (P < 0.05, n = 5), which was inhibited by 5-HD. Isoflurane caused a dose-dependent rightward shift in ATP inhibition of mitO[K.sub.ATP] channels, which increased the I[C.sub.50] for ATP from 335 [+ or -] 4 to 940 [+ or -] 34 [micro]M at 0.8 mM (P < 0.05, n = 5-8). We conclude that direct activation of the mitO[K.sub.ATP] channel by isoflurane is likely to contribute to volatile anesthetic-induced myocardial preconditioning. heart; mitochondria; potassium channel; volatile anesthetics

Delayed cardioprotection by isuflurane: role of [K.sub.ATP] channels. Am J Physiol Heart Circ Physiol 283: H61-H68, 2002. First published March 7, 2002; 10.1152/ ajpheart.01040.2001.--Isoflurane mimics the cardioprotective effect of acute ischemic preconditioning with an acute memory phase. We determined whether isoflurane can induce delayed cardioprotection, the involvement of ATP-sensitive potassium ([K.sub.ATP]) channels, and cellular location of the channels. Neonatal New Zealand White rabbits at 7-10 days of age (n = 5-16/group) were exposed to 1% isoflurane-100% oxygen for 2 h. Hearts exposed 2 h to 100% oxygen served as untreated controls. Twenty-four hours later resistance to myocardial ischemia was determined using an isolated perfused heart model. Isoflurane significantly reduced infarct size/area at risk (means [+ or -] SD) by 50% (10 [+ or -] 5%) versus untreated controls (20 [+ or -] 6%). Isoflurane increased recovery of preischemic left ventricular developed pressure by 28% (69 [+ or -] 4%) versus untreated controls (54 [+ or -] 6%). The mitochondrial [K.sub.ATP] channel blocker 5-hydroxydecanoate (5-HD) completely (55 [+ or -] 3%) and the sarcolemmal [K.sub.ATP] channel blocker HMR 1098 partially (62 [+ or -] 3%) attenuated the cardioprotective effects of isoflurane. The combination of 5-HD and HMR-1098 completely abolished the cardioprotective effect of isoflurane (56 [+ or -] 5%). We conclude that both mitochondrial and sarcolemmal [K.sub.ATP] channels contribute to isoflurane-induced delayed cardioprotection. ischemia; heart; infarct size; volatile anesthetics; myocardial preconditioning

Researchers have investigated the impact of sevoflurane, a halogenated ether and strong inhalational anesthetic, on the inward rectifier potassium channel current (Ikir) in guinea pig cardiac ventricular cells. This work was undertaken using the whole cell patch-clamp methodology. It was possible to establish that the sevoflurane interaction site with the inward rectifier potassium channel is an electrical distance of 0.2 from the extracellular side.

Halothane-anesthetized rats were analyzed after the administration of exogenous NO to determine the effects of neuronal NO synthase (nNOS) on cerebral blood flow (CBF) regulation during hypercapnia. Administration of NO in anesthetized rats during hypercapnia enhanced cerebrocortical vasodilation. Inhibition of nNOS by 7-nitroindazole impaired CBF response to hypercapnia indicating the role of nNOS in modulating CBF response during hypercapnia.