Isoflurane Anesthesia Research Articles

Evaluating the effects of three anesthetic regimens on retinal structure and function in the living mouse eye using a customized bimodal OCT/ORG system

Abstract Optical coherence tomography (OCT) is a non-invasive imaging technique that generates cross-sectional images of biological tissues at millimeter-level penetration depth with micron-level resolution. In this study, we developed a custom spectral-domain OCT system equipped with a flash stimulus module for dual-modality imaging of mouse retinal structure and function. The axial resolution of the system reached ∼2.7/2 μm in air/tissue following optimization and calibration. We deployed this system to conduct in vivo OCT structural imaging and optoretinography (ORG) functional assessment of mouse retina to investigate the effects of three anesthesia regimens. Statistical analysis of the results indicates that anesthesia results in a smaller relative intensity of inner/outer segment junction and external limiting membrane and a thickening of the outer segment compared to an awake state. Meanwhile, significant differences were observed in the effects on retinal structure and function between isoflurane and tribromoethanol anesthesia, while differences between isoflurane and esketamine+xylazine groups were small. These findings suggest that the type of anesthetic should be taken into account when comparing imaging results across studies reported in the literature.

Clinical effects of vatinoxan co-administered with medetomidine and methadone in healthy dogs undergoing isoflurane anaesthesia

Clinical effects of vatinoxan co-administered with medetomidine and methadone in healthy dogs undergoing isoflurane anaesthesia

Comparison of the Competitiveness for Danish, Dutch, and German Piglet Producers under Consideration of Country-Specific Methods of Piglet Castration with Anesthesia

Pig producers in Europe adopt different production methods for male pig fattening. More than half of the animals are surgically castrated. The different interpretations of animal welfare in different countries lead to market differentiation and economically different production conditions, which do not restrict trade, but economically lead to drastic competitive changes for local producers. While the Netherlands has already implemented surgical castration for the export market, using CO2 narcosis (NL), Denmark and Germany are each introducing their strategies with local anesthesia (DK) and isoflurane anesthesia (DE), respectively. Using typical pig farms from the agri benchmark Pig Network, the additional costs and economic impacts of animal welfare regulations are calculated. In Germany, isoflurane anesthesia increases costs by EUR 28.54 to EUR 49.86 per sow, or EUR 1.93 to EUR 3.81 per male piglet. This corresponds to a cost increase of around 5% per piglet. In Denmark, the costs of local anesthesia with procaine increase more moderately by EUR 3.55 to EUR 5.05 per sow, or around EUR 0.30 per male piglet. The cost increase here is less than 1% per piglet. The additional costs are leading to a loss of competitiveness for Denmark and Germany compared to the Netherlands. However, Germany is also losing profit significantly compared to Denmark. This study highlights that animal welfare regulations can negatively impact the competitiveness of the pig sector. It emphasizes the need for countries to implement such regulations carefully, ensuring that they do not lead to the loss of production or international competitiveness. A balanced approach that supports both animal welfare and economic sustainability is essential.

Retrospective evaluation of the impact of atropine administration on incidence of post-operative colic in healthy, isoflurane-anaesthetised horses.

In anaesthetised horses, bradycardia secondary to high vagal tone can reduce cardiac output and blood pressure. The use of anticholinergics in horses is limited due to concerns about ileus and abdominal discomfort. This retrospective study sought to determine the prevalence of post-operative abdominal discomfort in healthy horses that received atropine under isoflurane anaesthesia. A retrospective evaluation of 222 general anaesthesia events between January 2019 and December 2019 was undertaken. One hundred and eleven horses that received atropine were identified, and 111 case match controls that did not receive atropine were also selected. Information gathered from the medical records included signalment, anaesthetic drugs, surgical procedures, duration of anaesthesia and surgery, dobutamine and atropine administration, and the occurrence of abdominal discomfort for 24 h after anaesthesia. After initial data analysis, a second cohort of records was assessed separately. The horses in this group were castrated under general anaesthesia (with or without atropine; n = 68). Logistic regression models and Fisher's exact tests were used to look for factors contributing to abdominal discomfort post-anaesthesia. The significance level was set to 5% (p < 0.05). Atropine administration was not associated with the development of post-anaesthetic abdominal discomfort (OR = 2.121, 95% CI [0.767, 5.869]; p = 0.2). Overall, 18/222 (8.1%) incidences of abdominal discomfort were identified. All incidents occurred in colts undergoing castration. In a separate analysis of only horses anaesthetised for castration, atropine was associated with developing abdominal discomfort (OR = 3.143, 95% CI [1.082, 9.132]; p = 0.04). Atropine was not associated with post-operative abdominal discomfort except in colts undergoing castration. All episodes of discomfort were mild and resolved with minimal intervention. The potential impact of insufficient analgesia in horses undergoing castration is a confounding factor and requires prospective investigation. Overall, atropine at a dose of 0.006 mg/kg IV appears to be a safe method to treat bradycardia in otherwise healthy horses anaesthetised for orthopaedic and upper airway procedures. Further work is required to determine if atropine is safe for colts undergoing castration.

GABAergic neurons in the central amygdala promote emergence from isoflurane anesthesia in mice.

Recent evidence indicates that general anesthesia and sleep-wake behavior share some overlapping neural substrates. GABAergic neurons in the central amygdala (CeA) have a high firing rate during wakefulness and play a role in regulating arousal-related behaviors. The objective of this study is to investigate whether CeA GABAergic neurons participate in the regulation of isoflurane general anesthesia and uncover the underlying neural circuitry. Fiber photometry recording was used to determine the changes in calcium signals of CeA GABAergic neurons during isoflurane anesthesia in Vgat-Cre mice. Chemogenetic and optogenetic approaches were used to manipulate the activity of CeA GABAergic neurons, and a righting reflex test was used to determine the induction and emergence from isoflurane anesthesia. Cortical electroencephalogram (EEG) recording was used to assess the changes in EEG spectral power and burst-suppression ratio during 0.8% and 1.4% isoflurane anesthesia, respectively. Both male and female mice were used in this study. The calcium signals of CeA GABAergic neurons decreased during the induction of isoflurane anesthesia and was restored during the emergence. Chemogenetic activation of CeA GABAergic neurons delayed induction time (mean ± SD, vehicle vs. clozapine-N-oxide: 58.75±5.42 s vs. 67.63±5.01 s; n=8, P=0.0017) and shortened emergence time (385.50±66.26 s vs. 214.60±40.21 s; n=8, P=0.0017) from isoflurane anesthesia. Optogenetic activation of CeA GABAergic neurons produced a similar effect. Furthermore, optogenetic activation decreased EEG delta power (Pre-stim vs. Stim: 46.63%±4.40% vs. 34.16%±6.47%; n=8, P=0.0195) and burst-suppression ratio (83.39%±5.15% vs. 52.60%±12.98%; n=8, P=0.0002). Moreover, optogenetic stimulation of terminals of CeA GABAergic neurons in the basal forebrain (BF) also promoted cortical activation and accelerated behavioral emergence from isoflurane anesthesia. Our results suggest that CeA GABAergic neurons play a role in general anesthesia regulation, which facilitates behavioral and cortical emergence from isoflurane anesthesia through the GABAergic CeA-BF pathway.

Safety of a Medicinal Product Based on Human Glial Progenitor Cells: A Pilot Study of Retrobulbar Administration in C57BL/6J Mice

INTRODUCTION. Stem cell therapy is a promising treatment method for various diseases and injuries, but its safety has yet to be determined. Therefore, studying the safety of administering a xenogeneic cell-based medicinal product (CBMP) into the retro-orbital venous sinus is essential for developing protocols for further studies of potential medicinal products for neurological conditions.AIM. The aim of the study was to determine the optimal dose of a CBMP derived from glial progenitor cells (GPCs) and to evaluate its safety during retrobulbar administration in C57BL/6J mice.MATERIALS AND METHODS. GPCs were derived from human induced pluripotent stem cells by stepwise differentiation and cultured in DMEM/F12 supplemented with epidermal growth factor and ciliary neurotrophic factor. Matrigel was used as a substrate. GPCs were injected into the retro-orbital venous sinus of male C57BL/6J mice under isoflurane anaesthesia once a week for two months. The study analysed changes in biochemical blood parameters and behaviour. The quantities of activated astrocytes and glial cells were determined by postmortem immunohistochemical staining.RESULTS. The administration of GPCs at a dose of 500×103 cells/mouse, which was selected using literature data, induced an increase in the plasma levels of ala nine aminotransferase and aspartate aminotransferase. This could indicate cell damage and the development of inflammatory reactions. At doses reduced to one-third the initial GPC concentration or lower, the biochemical blood parameters of the treatment groups did not differ significantly from those of the control group. There were no significant differences in neuroinflammatory markers between the groups receiving GPCs at different doses, except for an increase in astrocyte activation at a dose of 150×103 cells/mouse, which could potentially indicate inflammatory processes in the brain. The study detected no pathological changes in the brain or cell damage markers in the blood of mice after retrobulbar GPC injections of 15×103 or 50×103 cells/mouse.CONCLUSIONS. The study results indicate that long-term therapy with GPCs is potentially safe for mice if the dose is optimal. The authors suggest using the optimal doses and the administration route established in this study for further research into the safety of intravenous administration of CBMPs for neurological conditions.

Electroencephalographic and Cardiovascular Assessments of Isoflurane-Anesthetized Dogs.

This study investigated the use of frontal electroencephalography (EEG) to monitor varying levels of isoflurane anesthesia in dogs. The patient state index (PSI), burst suppression ratio (SR), and waveforms, were continuously recorded while mean arterial blood pressure (MBP), heart rate, responses to electric stimuli, and subjective anesthetic "depth" were assessed every 3 min. At deep anesthesia (2.5× MAC - 3.2%), the PSI (6.5 ± 10.8) and MBP (45.6 ± 16.4 mmHg) were the lowest, and SR was the highest (78.3 ± 24.0%). At 1× MAC (1.3%), the PSI and MBP increased significantly to 47.8 ± 12.6 and 99.8 ± 13.2, respectively, and SR decreased to 0.5 ± 2.5%. The EEG was predominantly isoelectric at 2×-2.5× MAC, indicating unconsciousness and unresponsiveness. As anesthesia lightened, waveforms transitioned to flatter and faster activity patterns with a response to noxious stimuli, suggesting regained consciousness. The PSI and MBP exhibited a stronger correlation (ρ = 0.8098, p = 0.001) than the relationship of PSI with heart rate (ρ = -0.2089, p = 0.249). Five of the six dogs experienced rough recovery, possibly due to high SR and low MBP. These findings suggest that EEG monitoring in dogs can be a valuable tool for the real-time tracking of brain states and can be used to guide the management of isoflurane anesthesia.

COMPARATIVE EVALUATION OF CARDIOVASCULAR PARAMETERS OF PROPOFOL / ETOMIDATE FOR ABDOMINAL SURGERIES IN CALVES

The calves presented with abdominal affections requiring surgical interventions were randomly divided into group I and group II with seven animals in each group. All the animals were premedicated with dexmedetomidine at 2 µg/kg body weight, pentazocine at 1 mg/kg body weight and midazolam at 0.2 mg/kg body weight intravenously. In group I and group II, anaesthetic induction was carried out with propofol at 2 mg/kg body weight and etomidate at 0.25 mg/kg body weight intravenously, respectively. Both the groups were maintained in Isoflurane anaesthesia with modified oro-endotracheal tube. The cardiovascular parameters such as heart rate, mean arterial blood pressure, systolic and diastolic blood pressure were monitored during different stages of anaesthesia in both the groups. In both the groups, a significant decrease in heart rate was noticed after premedication and anaesthetic induction. A Significant increase was noticed in systolic blood pressure values after anaesthetic induction and 10th minute of anaesthesia in group I and a highly significant increase was noticed after induction up to 10 minutes in group II. A highly significant increase in diastolic blood pressure was noticed after induction and 10th minute in group I, whereas significant increase was noticed in mean arterial pressure after induction and 15th minute of anaesthesia in group II animals. The cardiovascular parameters recorded in the present study concluded etomidate produced better cardiovascular stability and minimal respiratory depression compared to propofol throughout the anaesthetic period.

Assessing overall oxidative metabolism of high-metabolic-rate abdominal organs in diet-induced obesity using compartmental modeling of [11C]acetate-PET

[11C]Acetate PET imaging is a powerful tool for visualizing and studying metabolic processes in various tissues and organs, particularly in the context of heart disease and prostate cancer. The tracer can reflect tissue perfusion, oxidative metabolism, and fatty acid synthesis[1]. Male Sprague Dawley rats on standard (SD n=6) and after 8-10 weeks on a high-fat diet (HFD n=6) were studied for [11C]acetate turnover. Longitudinal utilization of the tracer in the abdomen was recorded using Siemens Inveon µ-PET/CT. Under isoflurane anesthesia, rats underwent 60 min dynamic [11C]acetate-PET scans and additionally on the next week venous blood sampling for [11C]CO2-metabolite analysis (7-time points; SD n=3; HFD n=3) accordingly[2]. Delineation of kidneys, myocardium and liver; SUV calculations; 1-tissue compartmental modeling with blood-to-plasma ratio and [11C]CO2-metabolite correction were performed in PMOD3.8; clearance rate was calculated in Carimas2.1. Semi-quantification methods demonstrated contrasting outcomes between the groups. Increased renal (right p=0.04, left p=0.03) and myocardial (p=0.01) uptake (Fig.A) was observed in the HFD group, while clearance rates in kidneys (right p=0.01, left p=0.04) and liver (p=0.03) were decreased (Fig.B). Blood sampling for [11C]CO2-metabolite analysis revealed differing profiles between the groups, with a maximum peak at 30 min in SD and two peaks at 5 and 40 min in HFD animals (Fig.C). Deeper analysis using 1-tissue compartmental modeling shed light on the hepatic uptake and showed a difference in the volume of distribution (Vt), which was significantly decreased in the HFD group (p&lt;0.001) (Fig.D). The study indicates [11C]acetate accumulation in metabolically active organs: kidneys, heart, and liver. Compartmental modeling demonstrated the necessity of metabolite correction for quantitative analysis. Previously, Sprague Dawley rats on HFD were studied for fatty acid uptake and decline of VLDL synthesis[3] that can correlate with the variation of Vt in the liver and confirm pathological changes in organ functioning. Please click on the 'PDF' for the full abstract!

18F]FDG Brain Imaging Assessment in Preclinical Acute and Chronic Stress Models Using Statistical Parametric Mapping

Previous studies have used [18F]FDG as a biomarker for depression, anxiety, or chronic stress in cancer patients1,2. However, they lack specific brain patterns and assessment in preclinical models, limiting their translational potential. Hence, we set out to establish preclinical [18F]FDG brain PET as a method to evaluate stress in vivo. Lean C57BL/6J mice were exposed to acute and chronic cooling, restraint, and surgical stress. Obese mice were on a high-fat diet for eight weeks and exposed to cooling and restraint stress. [18F]FDG was injected into fasted animals under brief isoflurane anesthesia via the tail vein. Following a 60-minute distribution phase during acute stress or one day after stress exposure (chronic), animals underwent static μPET/CT scans (Siemens® Inveon). Brain images were spatially normalized, and intensity normalization was performed using proportional and histogram-based scaling3 with PMOD (V. 3.8). Normalized images were analyzed using a brain atlas (M. Mirrione) to extract regional SUV and statistical parametric mapping (SPM12) for voxel-wise comparison. Overall, in lean mice, total brain [18F]FDG-uptake was significantly reduced across all stress models except for acute restraint, whereas total uptake was unaffected in obese mice. Subsequent brain atlas analysis of lean mice revealed changes in regional uptake for all stressors except for chronic cooling, while obese mice were affected by only acute and chronic restraint. Using SPM, we validated stress-specific hypo- and hypermetabolic areas in the brains of lean mice upon acute cooling, acute and chronic restraint, and surgery and in obese mice after chronic restraint (see Figure). We also identified overlapping areas in the brain stem and hypothalamus region following acute cooling and surgery, possibly due to post-surgical hypothermia. Our findings show that static [18F]FDG-imaging is a valuable tool for noninvasively assessing brain activity in a multitude of stress models and a suitable translational tool for stress research in mice. Please click on the 'PDF' for the full abstract!

Mouse aversion to induction with isoflurane using the drop method.

Isoflurane anesthesia prior to carbon dioxide euthanasia is recognized as a refinement by many guidelines. Facilities lacking access to a vaporizer can use the "drop" method, whereby liquid anesthetic is introduced into an induction chamber. Knowing the least aversive concentration of isoflurane is critical. Previous work has demonstrated that isoflurane administered with the drop method at a concentration of 5% is aversive to mice. Other work has shown that lower concentrations (1.7% to 3.7%) of isoflurane can be used to anesthetize mice with the drop method, but aversion to these concentrations has not been tested. We assessed aversion to these lower isoflurane concentrations administered with the drop method, using a conditioned place aversion (CPA) paradigm. Female C57BL/6J (OT-1) mice (n = 28) were randomly allocated to one of three isoflurane concentrations: 1.7%, 2.7%, and 3.7%. Mice were acclimated to a light-dark apparatus. Prior to and following dark (+ isoflurane) and light chamber conditioning sessions, mice underwent an initial and final preference assessment; the change in the duration spent within the dark chamber between the initial and final preference tests was used to calculate a CPA score. Aversion increased with increasing isoflurane concentration: from 1.7% to 2.7% to 3.7% isoflurane, mean ± SE CPA score decreased from 19.6 ± 20.1 s to -25.6 ± 23.2 s, to -116.9 ± 30.6 s (F1,54 = 15.4, p < 0.001). Our results suggest that, when using the drop method to administer isoflurane, concentrations between 1.7% and 2.7% can be used to minimize female mouse aversion to induction.

Effects of passive smoking on cortical spreading depolarization in male and female mice

BackgroundPatients with migraine are typically advised to avoid passive smoking because it may aggravate headaches and other health conditions. However, there is insufficient high-quality evidence on the association between passive smoking and migraine, which warrants further investigation using animal models. Therefore, using a mouse model, we examined the effect of passive smoking on susceptibility to cortical spreading depolarization (CSD), the biological basis of migraine with aura.FindingsFifty C57BL/6 mice (25 males and 25 females) were exposed for one hour to cigarette smoke or room air. Subsequently, potassium chloride (KCl) was administered under isoflurane anesthesia to induce CSD, and the CSD threshold, frequency of induction, and propagation velocity were determined. The threshold to induce CSD (median [interquartile range (IQR)]) was significantly lower in female mice (adjusted p = 0.01) in the smoking group (0.05 [0.05, 0.088]) than in the sham group (0.125 [0.1, 0.15]); however, there was no significant difference in the male mice (adjusted p = 0.77). CSD frequency or propagation velocity did not differ significantly between the two groups for either sex.ConclusionsFemale mice in the smoking group showed lower CSD threshold compared to the sham group, suggesting a potential sex-specific difference in the effect of smoking on the pathogenesis of CSD and migraine with aura. This finding may contribute to the understanding of migraine pathophysiology in association with passive smoking and sex difference.

Isoflurane anesthesia decreases excitability of inhibitory neurons in the basolateral amygdala leading to anxiety‑like behavior in aged mice.

Anxiety after surgery can be a major factor leading to postoperative cognitive dysfunction, particularly in elderly patients. The role of inhibitory neurons in the basolateral amygdala (BLA) in anxiety-like behaviors in aged mice following isoflurane anesthesia remains unclear. Therefore, the present study aimed to investigate the role of inhibitory neurons in isoflurane-treated mice. A total of 30 C57BL/6 mice (age, 13 months) were allocated into the control and isoflurane anesthesia groups (15 mice/group) and were then subjected to several neurological assessments. Behavioral testing using an elevated plus maze test showed that aged mice in the isoflurane anesthesia group displayed significant anxiety-like behavior, since they spent more time in the closed arm, exhibited more wall climbing behavior and covered more distance. In addition, whole-cell patch-clamp recording revealed that the excitability of the BLA excitatory neurons was notably increased following mice anesthesia with isoflurane, while that of inhibitory neurons was markedly reduced. Following mice treatment with diazepam, the excitability of the BLA inhibitory neurons was notably increased compared with that of the excitatory neurons, which was significantly attenuated. Overall, the results of the current study indicated that anxiety-like behavior could occur in aged mice after isoflurane anesthesia, which could be caused by a reduced excitability of the inhibitory neurons in the BLA area. This process could enhance excitatory neuronal activity in aged mice, thus ultimately promoting the onset of anxiety-like behaviors.

Cardiorespiratory effects of Gamma-Hydroxybutyric Acid (GHB) during isoflurane anaesthesia in pigs

ObjectiveTo investigate the haemodynamic effects of gamma-hydroxybutyric acid (GHB) in isoflurane anaesthetised pigs. Study DesignExperimental, randomized, non-blinded, cross-over study. AnimalsA group of six stress-resistant Landrace pigs (approximately 3 months old; three male, three female; bodyweight 39.2 ± 4 kg (mean ± standard deviation). MethodsAfter premedication (midazolam 0.5 mg kg-1 and ketamine 10 mg kg-1 intramuscularly), and induction [propofol 0.25–0.5 mg kg-1 intravenously IV)], anaesthesia was maintained with isoflurane in oxygen, and either GHB 250 mg kg-1 IV or an equal volume of saline was administered (minimum washout period 1 week). Systolic (SAP), diastolic (DAP), and mean (MAP) arterial pressures, heart rate and rhythm, respiratory rate, were recorded every 5 minutes for 2 hours. Arterial samples were collected for blood gas and pharmacokinetic analyses. Relative changes from baseline were calculated and compared between treatments using a mixed model with time, period, and treatment as variables (α < 0.05). ResultsChanges from baseline differed significantly between treatments (p < 0.001) for SAP (GHB -1.6 ± 10.7; saline -5.9 ± 14.8 mmHg), DAP (GHB +2.9 ± 9.6; saline -6.5 ± 10.7 mmHg) and MAP (GHB +2.2 ± 10.5; saline -5.7 ± 9.6 mmHg). Statistical analysis of secondary outcomes suggested effects on PaO2 (GHB -45.2 ± 29.8 mmHg [-6.03 ± 3.97 kPa]; saline +24.5 ± 32.4 mmHg [+3.27 ± 4.32 kPa]; p < 0.001) and PaCO2 (GHB -2 ± 10 mmHg [-0.27 ± 1.33 kPa]; saline -9 ± 8 mmHg [-1.20 ± 1.07 kPa]; p < 0.001). Mean maximum plasma concentration of GHB was 1171.1 ± 229.3 μg mL-1, with volume of distribution 335.3 ± 68.5 mL kg-1, clearance 77.2 ± 19.12 mL kg-1 hour-1 and elimination half-life 3.10 ± 0.80 hours. Conclusions and clinical relevanceGHB did not cause severe physiological side effects and may reduce cardiovascular depression.

MEF2C Alleviates Postoperative Cognitive Dysfunction by Repressing Ferroptosis.

Ferroptosis, a form of programmed cell death featured by lipid peroxidation, has been proposed as a potential etiology for postoperative cognitive dysfunction (POCD). Myocyte-specific enhancer factor 2C (MEF2C), a transcription factor expressed in various brain cell types, has been implicated in cognitive disorders. This study sought to ascertain whether MEF2C governs postoperative cognitive capacity by affecting ferroptosis. Transcriptomic analysis of public data was used to identify MEF2C as a candidate differentially expressed gene in the hippocampus of POCD mice. The POCD mouse model was established via aseptic laparotomy under isoflurane anesthesia after treatment with recombinant adeno-associated virus 9 (AAV9)-mediated overexpression of MEF2C and/or the glutathione peroxidase 4 (GPX4) inhibitor RSL3. Cognitive performance, Nissl staining, and ferroptosis-related parameters were assessed. Dual-luciferase reporter gene assays and chromatin immunoprecipitation assays were implemented to elucidate the mechanism by which MEF2C transcriptionally activates GPX4. MEF2C mRNA and protein levels decreased in the mouse hippocampus following anesthesia and surgery. MEF2C overexpression ameliorated postoperative memory decline, hindered lipid peroxidation and iron accumulation, and enhanced antioxidant capacity, which were reversed by RSL3. Additionally, MEF2C was found to directly bind to the Gpx4 promoter and activate its transcription. Our findings suggest that MEF2C may be a promising therapeutic target for POCD through its negative modulation of ferroptosis.

Novel ryanodine receptor 1 (RYR1) missense gene variants in 2 pet dogs with fatal malignant hyperthermia identified by next-generation sequencing

ObjectiveEvaluate a precision medicine approach to confirm a tentative diagnosis of fatal malignant hyperthermia (MH) in isoflurane anesthetized pet dogs by identifying novel risk variants in known MH susceptibility genes. Study designRetrospective case series. AnimalsA 7-year-old male pitbull mix (Case #1), a 12-month-old male golden retriever (Case #2), and the dam and sire of Case #2. MethodsAvailable case histories and medical records were reviewed. Missense variants in MH susceptibility genes RYR-1, CACNA1S, and STAC3 (Case #2 only) were identified by next-generation sequencing of DNA from each case and the parents of Case #2 with confirmation by Sanger sequencing. The pathogenicity of variants was evaluated by multiple in silico approaches. ResultsBoth cases demonstrated clinical signs during isoflurane anesthesia consistent with volatile anesthetic induced MH, including tachypnea, tachycardia, severe hyperthermia and muscle rigidity. Despite whole body cooling and other treatments, both dogs died after cardiac arrest within 15 minutes of detecting hyperthermia. Gene sequencing identified novel missense RYR-1 variants in Case #1 (p.Gly2375Arg) and Case #2 (p.Pro152Leu). Both variants were likely pathogenic based on multiple criteria, including gene location, amino acid alteration, and population allele frequency. The Case #1 variant was identical to a known human diagnostic MH variant (p.Gly2375Arg). Neither parent of Case #2 had the Case #2 variant, indicating this variant was not inherited, but arose de novo in a germ cell of either parent, or early in embryogenesis. Whole genome sequence analysis confirmed parentage. Two missense variants were identified in the CACNA1S gene. Both variants were considered nonpathogenic. No variants were identified in STAC3. Conclusions and clinical relevanceLike humans, MH susceptibility in dogs is associated with different rare variants located in pathogenic hotspots in the RYR-1 gene. Next-generation sequencing is a useful tool to assist in the definitive diagnosis of MH in dogs.

Local wakefulness-like activity of layer 5 cortex under general anaesthesia.

Consciousness, defined as being aware of and responsive to one's surroundings, is characteristic of normal waking life and typically is lost during sleep and general anaesthesia. The traditional view of consciousness as a global brain state has evolved toward a more sophisticated interplay between global and local states, with the presence of local sleep in the awake brain and local wakefulness in the sleeping brain. However, this interplay is not clear for general anaesthesia, where loss of consciousness was recently suggested to be associated with a global state of brain-widesynchrony that selectively involves layer 5 cortical pyramidal neurons across sensory, motor and associative areas. According to this global view, local wakefulness of layer 5 cortex should be incompatible with deep anaesthesia, a hypothesis that deserves to be scrutinised with causal manipulations. Here, we show that unilateral chemogenetic activation of layer 5 pyramidal neurons in the sensorimotor cortex of isoflurane-anaesthetised mice induces a local state transition from slow-wave activity to tonic firing in the transfected hemisphere. This wakefulness-like activity dramatically disrupts layer 5 interhemispheric synchrony with mirror-image locations in the contralateral hemisphere, but does not reduce the level of unconsciousness under deep anaesthesia, nor in the transitions to/from anaesthesia. Global layer 5synchrony may thus be a sufficient condition for anaesthesia-induced unconsciousness, but is not a necessary one, at least under isoflurane anaesthesia. Local wakefulness-like activity of layer 5 cortex can be induced and maintained under deep anaesthesia, encouraging further investigation into the local vs. global aspects of anaesthesia-induced unconsciousness. KEY POINTS: The neural correlates of consciousness have evolved from global brain states to a nuanced interplay between global and local states, evident in terms of local sleep in awake brains and local wakefulness in sleeping brains. The concept of local wakefulness remains unclear for general anaesthesia, where the loss of consciousness has been recently suggested to involve brain-wide synchrony of layer 5 cortical neurons. We found that local wakefulness-like activity of layer 5 cortical can be chemogenetically induced in anaesthetised mice without affecting the depth of anaesthesia or the transitions to and from unconsciousness. Global layer 5synchrony may thus be a sufficient but not necessary feature for the unconsciousness induced by general anaesthesia. Local wakefulness-like activity of layer 5 neurons is compatible with general anaesthesia, thus promoting further investigation into the local vs. global aspects of anaesthesia-induced unconsciousness.

Zona Incerta GABAergic Neurons Facilitate Emergence from Isoflurane Anesthesia in Mice.

The zona incerta (ZI) predominantly consists of gamma-aminobutyric acid (GABAergic) neurons, located adjacent to the lateral hypothalamus. GABA, acting on GABAA receptors, serves as a crucial neuromodulator in the initiation and maintenance of general anesthesia. In this study, we aimed to investigate the involvement of ZI GABAergic neurons in the general anesthesia process. Utilizing in-vivo calcium signal optical fiber recording, we observed a decrease in the activity of ZI GABAergic neurons during isoflurane anesthesia, followed by a significant increase during the recovery phase. Subsequently, we selectively ablated ZI GABAergic neurons to explore their role in general anesthesia, revealing no impact on the induction of isoflurane anesthesia but a prolonged recovery time, accompanied by a reduction in delta-band power in mice under isoflurane anesthesia. Finally, through optogenetic activation/inhibition of ZI GABAergic neurons during isoflurane anesthesia, we discovered that activation of these neurons facilitated emergence without affecting the induction process, while inhibition delayed emergence, leading to fluctuations in delta band activity. In summary, these findings highlight the involvement of ZI GABAergic neurons in modulating the emergence of isoflurane anesthesia.

Similarity and characterization of structural and functional neural connections within species under isoflurane anesthesia in the common marmoset

The common marmoset is an essential model for understanding social cognition and neurodegenerative diseases. This study explored the structural and functional brain connectivity in a marmoset under isoflurane anesthesia, aiming to statistically overcome the effects of high inter-individual variability and noise-related confounds such as physiological noise, ensuring robust and reliable data. Similarities and differences in individual subject data, including assessments of functional and structural brain connectivities derived from resting-state functional MRI and diffusion tensor imaging were meticulously captured. The findings highlighted the high consistency of structural neural connections within the species, indicating a stable neural architecture, while functional connectivity under anesthesia displayed considerable variability. Through independent component and dual regression analyses, several distinct brain connectivities were identified, elucidating their characteristics under anesthesia. Insights into the structural and functional features of the marmoset brain from this study affirm its value as a neuroscience research model, promising advancements in the field through fundamental and translational studies.

The impact of isoflurane anesthesia on brain metabolism in mice: An MRI and electroencephalography study.

Isoflurane is one of the most widely used anesthetic agents in rodent imaging studies. However, the impact of isoflurane on brain metabolism has not been fully characterized to date, primarily due to a scarcity of noninvasive technologies to quantitatively measure the brain's metabolic rate in vivo. In this study, using noncontrast MRI techniques, we dynamically measured cerebral metabolic rate of oxygen (CMRO2) under varying doses of isoflurane anesthesia in mice. Concurrently, systemic parameters of heart and respiration rates were recorded alongside CMRO2. Additionally, electroencephalogram (EEG) recording was used to identify changes in neuronal activities under the same anesthetic regimen employed in the MRI experiments. We found suppression of the CMRO2 by isoflurane in a dose-dependent manner, concomitant with a diminished high-frequency EEG activity. The degree of metabolic suppression by isoflurane was strongly correlated with the respiration rate, which offers a potential approach to calibrate CMRO2 measurements. Furthermore, the metabolic level associated with neural responses of the somatosensory and motor cortices in mice was estimated as 308.2μmol/100 g/min. These findings may facilitate the integration of metabolic parameters into future studies involving animal disease models and anesthesia usage.