Neuromuscular Weakness and Paralysis Produced by Snakebite Envenoming: Mechanisms and Proposed Standards for Clinical Assessment.

Respiratory and airway-protective muscle weakness caused by the blockade of neuromuscular transmission is a major cause of early mortality from snakebite envenoming (SBE). Once weakness is manifest, antivenom appears to be of limited effectiveness in improving neuromuscular function. Herein, we review the topic of venom-induced neuromuscular blockade and consider the utility of adopting clinical management methods originally developed for the safe use of neuromuscular blocking agents by anesthesiologists in operating rooms and critical care units. Failure to quantify neuromuscular weakness in SBE is predicted to cause the same significant morbidity that is associated with failure to do so in the context of using a clinical neuromuscular block in surgery and critical care. The quantitative monitoring of a neuromuscular block, and an understanding of its neurophysiological characteristics, enables an objective measurement of weakness that may otherwise be overlooked by traditional clinical examination at the bedside. This is important for the initial assessment and the monitoring of recovery from neurotoxic envenoming. Adopting these methods will also be critical to the conduct of future clinical trials of toxin-inhibiting drugs and antivenoms being tested for the reversal of venom-induced neuromuscular block.

A novel approach for the detection of cognitive impairment and delirium risk in older patients undergoing spine surgery.

BACKGROUND: Postoperative delirium is a common postsurgical complication in older patients and is associated with high morbidity and mortality. The objective of this study was to determine whether a digital cognitive assessment and patient characteristics could identify those at-risk. METHODS: Patients 65 years and older undergoing spine surgeries ≥3 h were evaluated as part of a single-center prospective observational cohort study at an academic medical center, from January 1, 2019, to December 31, 2020. Of 220 eligible patients, 161 were enrolled and 152 completed the study. The primary outcome of postoperative delirium was measured by the Confusion Assessment Method for the Intensive Care Unit or the Nursing Delirium Screening Scale, administered by trained nursing staff independent from the study protocol. Baseline cognitive impairment was identified using the tablet-based TabCAT Brain Health Assessment. RESULTS: Of the 152 patients included in this study, 46% were women. The mean [SD] age was 72 [5.4] years. Baseline cognitive impairment was identified in 38% of participants, and 26% had postoperative delirium. In multivariable analysis, impaired Brain Health Assessment Cognitive Score (OR 2.45; 95% CI, 1.05-5.67; p = 0.037), depression (OR 4.54; 95% CI, 1.73-11.89; p = 0.002), and higher surgical complexity Tier 4 (OR 5.88; 95% CI, 1.55-22.26; p = 0.009) were associated with postoperative delirium. The multivariate model was 72% accurate for predicting postoperative delirium, compared to 45% for the electronic medical record-based risk stratification model currently in use. CONCLUSION: In this prospective cohort study of spine surgery patients, age, cognitive impairment, depression, and surgical complexity identified patients at high risk for postoperative delirium. Integration of scalable digital assessments into preoperative workflows could identify high-risk patients, automate decision support for timely interventions that can improve patient outcomes and lower hospital costs, and provide a baseline cognitive assessment to monitor for postoperative cognitive change.

Neuroanatomical Basis for the Orexinergic Modulation of Anesthesia Arousal and Pain Control.

Hypothalamic orexin (hypocretin) neurons play crucial roles in arousal control. Their involvement in anesthesia and analgesia remains to be better understood. In order to enhance our view on the neuroanatomy, we systematically mapped the projections of orexin neurons with confocal microscope and light sheet microscope. We specifically expressed optogenetic opsins tagged with fluorescence markers in orexin neurons through adeno-associated viral infection in the mouse brain. The imaging results revealed fine details and novel features of the orexin projections throughout the brain, particularly related to the nuclei regulating arousal and pain. We then optogenetically activated orexin neurons in the lateral hypothalamus to study the effects on anesthesia-related behaviors. cFos staining showed that optogenetic stimulation can activate orexin neurons in the ChR2-mCherry group, but not the control mCherry group (62.86 ± 3.923% vs. 7.9 ± 2.072%; P < 0.0001). In behavior assays, optogenetic stimulation in the ChR2-mCherry group consistently elicited robust arousal from light isoflurane anesthesia (9.429 ± 3.804 s vs. 238.2 ± 17.42 s; P < 0.0001), shortened the emergence time after deep isoflurane anesthesia (109.5 ± 13.59 s vs. 213.8 ± 21.77 s; P = 0.0023), and increased the paw withdrawal latency in a hotplate test (11.45 ± 1.185 s vs. 8.767 ± 0.7775; P = 0.0317). The structural details of orexin fibers established the neuroanatomic basis for studying the role of orexin in anesthesia and analgesia.

Activation of orexin system facilitates anesthesia emergence and pain control.

Orexin (also known as hypocretin) neurons in the hypothalamus play an essential role in sleep-wake control, feeding, reward, and energy homeostasis. The likelihood of anesthesia and sleep sharing common pathways notwithstanding, it is important to understand the processes underlying emergence from anesthesia. In this study, we investigated the role of the orexin system in anesthesia emergence, by specifically activating orexin neurons utilizing the designer receptors exclusively activated by designer drugs (DREADD) chemogenetic approach. With injection of adeno-associated virus into the orexin-Cre transgenic mouse brain, we expressed the DREADD receptor hM3Dq specifically in orexin neurons and applied the hM3Dq ligand clozapine to activate orexin neurons. We monitored orexin neuronal activities by c-Fos staining and whole-cell patch-clamp recording and examined the consequence of orexin neuronal activation via EEG recording. Our results revealed that the orexin-DREADD mice with activated orexin neurons emerged from anesthesia with significantly shorter latency than the control mice. As an indication of reduced pain sensitivity, these orexin-DREADD mice took longer to respond to the 55 °C thermal stimuli in the hot plate test and exhibited significantly less frequent licking of the formalin-injected paw in the formalin test. Our study suggests that approaches to activate the orexin system can be beneficial in postoperative recovery.

Increased Cytokines at High Altitude: Lack of Effect of Ibuprofen on Acute Mountain Sickness, Physiological Variables, or Cytokine Levels.

Lundeberg, Jenny, John R. Feiner, Andrew Schober, Jeffrey W. Sall, Helge Eilers, and Philip E. Bickler. Increased cytokines at high altitude: lack of effect of ibuprofen on acute mountain sickness, physiological variables or cytokine levels. High Alt Med Biol. 19:249-258, 2018. INTRODUCTION: There is no consensus on the role of inflammation in high-altitude acclimatization. AIMS: To determine the effects of a nonsteroidal anti-inflammatory drug (ibuprofen 400 mg every 8 hours) on blood cytokines, acclimatization, acute mountain sickness (AMS, Lake Louise Score), and noninvasive oxygenation in brain and muscle in healthy volunteers. MATERIALS AND METHODS: In this double-blind study, 20 volunteers were randomized to receive ibuprofen or placebo at sea level and for 48 hours at 3800 m altitude. Arterial, brain, and leg muscle saturation with near infrared spectroscopy, pulse oximetry, and heart rate were measured. Blood samples were collected for cytokine levels and cytokine gene expression. RESULTS: All of the placebo subjects and 8 of 11 ibuprofen subjects developed AMS at altitude (p = 0.22, comparing placebo and ibuprofen). On arrival at altitude, the oxygen saturation as measured by pulse oximetry (SpO2) was 84.5% ± 5.4% (mean ± standard deviation). Increase in blood interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels occurred comparably in the placebo and ibuprofen groups (all not significant, univariate test by Wilcoxon rank sum). Increased IL-6 was associated with higher AMS scores (p = 0.002 by Spearman rank correlation). However, we found no difference or association in AMS score and blood or tissue oxygenation between the ibuprofen and placebo groups. CONCLUSIONS: We found that ibuprofen, at the package-recommended adult dose, did not have a significant effect on altitude-related increases in cytokines, AMS scores, blood, or tissue oxygenation in a population of healthy subjects with a high incidence of AMS.

Anesthetic protection of neurons injured by hypothermia and rewarming: roles of intracellular Ca2+ and excitotoxicity.

BACKGROUND: Mild hypothermia is neuroprotective after cerebral ischemia but surgery involving profound hypothermia (PH, temperature less than 18°C) is associated with neurologic complications. Rewarming (RW) from PH injures hippocampal neurons by glutamate excitotoxicity, N-methyl-D-aspartate receptors, and intracellular calcium. Because neurons are protected from hypoxia-ischemia by anesthetic agents that inhibit N-methyl-D-aspartic acid receptors, we tested whether anesthetics protect neurons from damage caused by PH/RW. METHODS: Organotypic cultures of rat hippocampus were used to model PH/RW injury, with hypothermia at 4°C followed by RW to 37°C and assessment of cell death 1 or 24 h later. Cell death and intracellular Ca were assessed with fluorescent dye imaging and histology. Anesthetic agents were present in the culture media during PH and RW or only RW. RESULTS: Injury to hippocampal CA1, CA3, and dentate neurons after PH and RW involved cell swelling, cell rupture, and adenosine triphosphate (ATP) loss; this injury was similar for 4 through 10 h of PH. Isoflurane (1% and 2%), sevoflurane (3%) and xenon (60%) reduced cell loss but propofol (3 µM) and pentobarbital (100 µM) did not. Isoflurane protection involved reduction in N-methyl-D-aspartate receptor-mediated Ca influx during RW but did not involve γ-amino butyric acid receptors or KATP channels. However, cell death increased over the next day. CONCLUSION: Anesthetic protection of neurons rewarmed from 4°C involves suppression of N-methyl-D-aspartate receptor-mediated Ca overload in neurons undergoing ATP loss and excitotoxicity. Unlike during hypoxia/ischemia, anesthetic agents acting predominantly on γ-aminobutyric acid receptors do not protect against PH/RW. The durability of anesthetic protection against cold injury may be limited.

Hypoxic preconditioning failure in aging hippocampal neurons: impaired gene expression and rescue with intracellular calcium chelation.

Hypoxic preconditioning in the brain (HPC), a phenomenon whereby noninjurious hypoxia induces resistance to cell death following ischemia, requires the expression of specific genes. Declines in signal transduction pathway activity with aging may decrease the genomic response to HPC and limit its neuroprotective efficacy. To test this, we determined how signal transduction gene expression, intracellular Ca(2+) levels, and phosphorylation of the survival-associated kinase Akt differ in hippocampal slice cultures (HSCs) made from postnatal day 7-10 (P7-10) and 2-year-old rats following HPC. HPC neuroprotection decreased with increasing source animal age, and HPC could not be demonstrated in HCSs made from animals >6 months of age, despite adjusting the duration of hypoxic exposure. Preconditioning protection required the survival kinase Akt in P10 hippocampal slices cultures. In P9 cultures, HPC increased Akt phosphorylation and the expression of prosurvival genes, including Bcl-2, heat shock proteins, protein kinases, c-jun, and NfκB. Lack of increased Akt phosphorylation and a greatly diminished signaling pathway gene response were found in HSCs from aging animals. Moderate and transient increases in [Ca(2+) ](i) during HPC occurred in P7-10 HSCs, but [Ca(2+) ](i) was persistently increased at 1 and 24 hr after preconditioning in HSCs from 2-year-old rats. The intracellular Ca(2+) chelator BAPTA-AM facilitated HPC neuroprotection in 2-year-old HSCs and restored the pattern of post-HPC gene expression seen in immature animals. We conclude that age-related loss of preconditioning may be due to altered intracellular Ca(2+) homeostasis (excess and sustained increase in [Ca(2+) ](i) ) and is a lesion that prevents critical elements of neuroprotective signal transduction.

Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen-glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss.

Oxygen-glucose deprivation (OGD) initiates a cascade of intracellular responses that culminates in cell death in sensitive species. Neurons from Arctic ground squirrels (AGS), a hibernating species, tolerate OGD in vitro and global ischemia in vivo independent of temperature or torpor. Regulation of energy stores and activation of mitogen-activated protein kinase (MAPK) signaling pathways can regulate neuronal survival. We used acute hippocampal slices to investigate the role of ATP stores and extracellular signal-regulated kinase (ERK)1/2 and Jun NH(2)-terminal kinase (JNK) MAPKs in promoting survival. Acute hippocampal slices from AGS tolerated 30 mins of OGD and showed a small but significant increase in cell death with 2 h OGD at 37 degrees C. This tolerance is independent of hibernation state or season. Neurons from AGS survive OGD despite rapid ATP depletion by 3 mins in interbout euthermic AGS and 10 mins in hibernating AGS. Oxygen-glucose deprivation does not induce JNK activation in AGS and baseline ERK1/2 and JNK activation is maintained even after drastic depletion of ATP. Surprisingly, inhibition of ERK1/2 or JNK during OGD had no effect on survival, whereas inhibition of JNK increased cell death during normoxia. Thus, protective mechanisms promoting tolerance to OGD by AGS are downstream from ATP loss and are independent of hibernation state or season. Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1307-1319; doi:10.1038/jcbfm.2008.20; published online 9 April 2008.

Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability.

The ability of fishes, amphibians, and reptiles to survive extremes of oxygen availability derives from a core triad of adaptations: profound metabolic suppression, tolerance of ionic and pH disturbances, and mechanisms for avoiding free-radical injury during reoxygenation. For long-term anoxic survival, enhanced storage of glycogen in critical tissues is also necessary. The diversity of body morphologies and habitats and the utilization of dormancy have resulted in a broad array of adaptations to hypoxia in lower vertebrates. For example, the most anoxia-tolerant vertebrates, painted turtles and crucian carp, meet the challenge of variable oxygen in fundamentally different ways: Turtles undergo near-suspended animation, whereas carp remain active and responsive in the absence of oxygen. Although the mechanisms of survival in both of these cases include large stores of glycogen and drastically decreased metabolism, other mechanisms, such as regulation of ion channels in excitable membranes, are apparently divergent. Common themes in the regulatory adjustments to hypoxia involve control of metabolism and ion channel conductance by protein phosphorylation. Tolerance of decreased energy charge and accumulating anaerobic end products as well as enhanced antioxidant defenses and regenerative capacities are also key to hypoxia survival in lower vertebrates.

The inhaled anesthetic, isoflurane, enhances Ca2+-dependent survival signaling in cortical neurons and modulates MAP kinases, apoptosis proteins and transcription factors during hypoxia.

We tested whether the protection of hypoxic neurons by the inhaled anesthetic isoflurane is related to the Ca2+-dependent phosphorylation of MAP kinases and anti-apoptotic co-factors. In cultures of mouse cortical neurons we measured changes in the phosphorylation of Ca2+-dependent and Ca2+-independent MAP kinases, transcription factors, and apoptosis regulators after hypoxia or hypoxia combined with isoflurane (1% in gas phase). In hypoxic neurons, isoflurane reduced cell death and TUNEL staining by >80%. Isoflurane released Ca2+ from intracellular stores, increasing [Ca2+]i in oxygenated neurons by approximately 20%. Neuroprotection was associated with a smaller increase in [Ca2+]i in hypoxic neurons and required IP3 receptors and phospholipase C. In hypoxic neurons, isoflurane increased the phosphorylation of the Ca2+-dependent MAP kinases Pyk2 and p42/44 (ERK). The Ca2+-independent MAP kinase p38 pathway showed increased phosphorylation with isoflurane but not with ionomycin, a Ca2+ ionophore. JNK was phosphorylated in hypoxic neurons in the presence of isoflurane, as was the transcription factor c-Jun; JNK inhibition with SP600125 prevented both phosphorylation of c-Jun and neuroprotection. Isoflurane decreased phosphorylation of the pro-apoptotic cofactors Bad and p90RSK and increased Akt phosphorylation. However, with the exception of c-Jun, transcription factors (Elk-1, GSK-3, Forkhead, p90RSK) decreased or remained unchanged. We conclude that isoflurane's protection of hypoxic cortical neurons involves signaling that includes changes in intracellular Ca2+ regulation, several MAP kinase pathways and modulation of apoptosis regulators.

Isoflurane preconditions hippocampal neurons against oxygen-glucose deprivation: role of intracellular Ca2+ and mitogen-activated protein kinase signaling.

BACKGROUND: Isoflurane preconditions neurons to improve tolerance of subsequent ischemia in both intact animal models and in in vitro preparations. The mechanisms for this protection remain largely undefined. Because isoflurane increases intracellular Ca2+ concentrations and Ca2+ is involved in many processes related to preconditioning, the authors hypothesized that isoflurane preconditions neurons via Ca2+-dependent processes involving the Ca2+- binding protein calmodulin and the mitogen-activated protein kinase-ERK pathway. METHODS: The authors used a preconditioning model in which organotypic cultures of rat hippocampus were exposed to 0.5-1.5% isoflurane for a 2-h period 24 h before an ischemia-like injury of oxygen-glucose deprivation. Survival of CA1, CA3, and dentate neurons was assessed 48 later, along with interval measurements of intracellular Ca2+ concentration (fura-2 fluorescence microscopy in CA1 neurons), mitogen-activated protein kinase p42/44, and the survival associated proteins Akt and GSK-3beta (in situ immunostaining and Western blots). RESULTS: Preconditioning with 0.5-1.5% isoflurane decreased neuron death in CA1 and CA3 regions of hippocampal slice cultures after oxygen-glucose deprivation. The preconditioning period was associated with an increase in basal intracellular Ca2+ concentration of 7-15%, which involved Ca2+ release from inositol triphosphate-sensitive stores in the endoplasmic reticulum, and transient phosphorylation of mitogen-activated protein kinase p42/44 and the survival-associated proteins Akt and GSK-3beta. Preconditioning protection was eliminated by the mitogen-activated extracellular kinase inhibitor U0126, which prevented phosphorylation of p44 during preconditioning, and by calmidazolium, which antagonizes the effects of Ca2+-bound calmodulin. CONCLUSIONS: Isoflurane, at clinical concentrations, preconditions neurons in hippocampal slice cultures by mechanisms that apparently involve release of Ca2+ from the endoplasmic reticulum, transient increases in intracellular Ca2+ concentration, the Ca2+ binding protein calmodulin, and phosphorylation of the mitogen-activated protein kinase p42/44.

Isoflurane neuroprotection in hypoxic hippocampal slice cultures involves increases in intracellular Ca2+ and mitogen-activated protein kinases.

BACKGROUND: The volatile anesthetic isoflurane reduces acute and delayed neuron death in vitro models of brain ischemia, an action that the authors hypothesize is related to moderate increases in intracellular calcium concentration ([Ca2+]i). Specifically, the authors propose that during hypoxia, moderate increases in [Ca2+]i in the presence of isoflurane stimulates the Ca2+-dependent phosphorylation of members of the mitogen-activated protein kinase (MAP) kinase Ras-Raf-MEK-ERK pathway that are critical for neuroprotective signaling and suppression of apoptosis. METHODS: Death of CA1, CA3, and dentate neurons in rat hippocampal slice cultures was assessed by propidium iodide fluorescence 48-72 h after 60-75 min of hypoxia. [Ca2+]i in CA1 neurons was measured with fura-2 and fura-2 FF. Concentrations of the survival-signaling proteins Ras, MEK, MAP kinase p42/44, and protein kinase B (Akt) were assessed by immunostaining, and specific inhibitors were used to ascertain the role of Ca2+ and MAP kinases in mediating survival. RESULTS: Isoflurane, 1%, decreased neuron death in CA1, CA3, and dentate gyrus neurons after 60 but not 75 min of hypoxia. Survival of CA1 neurons required an inositol triphosphate receptor-dependent increase in [Ca2+]i of 30-100 nm that activated the Ras-Raf-MEK-ERK (p44/42) signaling pathway. Isoflurane also increased the phosphorylation of Akt during hypoxia. CONCLUSIONS: Isoflurane stimulates the phosphorylation of survival signaling proteins in hypoxic neurons. The mechanism involves a moderate increase in [Ca2+]i from release of Ca from inositol triphosphate receptor-dependent intracellular stores. The increase in [Ca2+]i sets in motion signaling via Ras and the MAP kinase p42/44 pathway and the antiapoptotic factor Akt. Isoflurane neuroprotection thus involves intracellular signaling well known to suppress both excitotoxic and apoptotic/delayed cell death.

Adaptive responses of vertebrate neurons to hypoxia.

The damage caused to mammalian neurons during ischaemic events in the brain (e.g. following a stroke), is an area of major interest to neuroscientists. The neurons of hypoxia-tolerant vertebrates offer unique models for identifying new strategies to enhance the survival of hypoxia-vulnerable neurons. In this review, we describe recent advances in our understanding of how hypoxia-tolerant neurons detect decreases in oxygen and create signals that have immediate and long-term effects on cell function and survival. Sensing and adapting to low oxygen tension involves numerous modalities with different times of activation and effect. Sensors include membrane proteins such as ionotropic ion channels, membrane or cytosolic heme proteins, mitochondrial proteins and/or oxygen sensitive transcription factors such as HIF-1alpha and NFkappaB. Signaling molecules involved in O(2) sensing include mitogen-activated protein kinases, ions such as Ca(2+) and metabolites such as adenosine. These signals act rapidly to reduce the conductance of ion channels (ion flux arrest) and production of energy (metabolic arrest), and slowly to activate specific genes. The ability to construct an energy budget, illustrating which physiological processes are depressed during both long-term and acute metabolic suppression in hypoxia-tolerant neurons, would be of significant value in devising new strategies for neuroprotection. Additionally it is not known how metabolism is regulated at 'pilot-light' levels at which energy-producing and energy-consuming processes are balanced. The regulation of organelle and cell fate during long-term hypoxia is almost completely unexplored, and whether programmed cell death and regeneration of lost neurons occur following protracted dormancy is also of considerable interest.

Molecular adaptations for survival during anoxia: lessons from lower vertebrates.

Anoxia-tolerant neurons from several species of animals may offer unparalleled opportunities to identify strategies that might be employed to enhance the hypoxia or ischemia tolerance of vulnerable neurons. In this review, the authors describe how the response of hypoxia-tolerant neurons to limited oxygen supply involves a suite of mechanisms that reduce energy expenditure in concert with decreased energy availability. This response avoids energy depletion, excitotoxic neuronal death, and apoptosis. Suppression of ion channel functions, particularly those of the ionotropic glutamate receptors, is a response common in hypoxia-tolerant neurons. The depression of excitability thereby achieved is essential given that the fundamental response to oxygen lack in anoxia-tolerant cells is a throttling down of metabolism to "pilot-light" levels. Many different types of processes have been found to down-regulate ion channel function. These include phosphorylation control, interactions with intracellular and extracellular ions, removal of active receptors from the neurolemma, and the direct sensing of oxygen by Na+ and K+ channels. Changes in [Ca2+]i may initiate a protective down-regulation of many different pumps or channels. Transcriptional events leading to differential and/or decreased expression of receptors, proteins, and their subunits are probably very important but little studied.