Predictors of first pass success without hypoxemia in trauma patients requiring emergent rapid sequence intubation.

OBJECTIVE: The predictors of first pass success (FPS) without hypoxemia among trauma patients requiring rapid sequence intubation (RSI) in the emergent setting are unknown. METHODS: Retrospective study of adult trauma patients requiring RSI during a 5-year period comparing the trauma patients achieving FPS without hypoxemia to those who did not. The primary outcome was FPS without hypoxemia evaluated by multivariate logistic regression adjusting for the neuromuscular blocking agent used (succinylcholine or rocuronium), hypoxemia prior to RSI, Glasgow Coma Scale (GCS) scores, the presence of head or facial trauma, and intubating operator level of training. RESULTS: 246 patients met our inclusion criteria. The overall FPS rate was 89%, and there was no statistical difference between those receiving either paralytic agent. 167 (69%) patients achieved FPS without hypoxemia. The two groups (those achieving FPS without hypoxemia and those who did not) had similar mean GCS, mean Injury Severity Scores, presence of head or facial trauma, the presence of penetrating trauma, intubating operator-level training, use of direct laryngoscopy, hypoxemia prior to RSI, heart rate per minute, mean systolic blood pressure, and respiratory rate. In the multivariate regression analysis, the use of succinylcholine and GCS score of 13-15 were found to have adjusted ORs of 2.1 (95% CI 1.2 to 3.8) and 2.0 (95% CI 1.0 to 3.3) for FPS without hypoxemia, respectively. CONCLUSION: Trauma patients requiring emergency department RSI with high GCS score and those who received succinylcholine had higher odds of achieving FPS without hypoxemia, a patient safety goal requiring more study. LEVEL OF EVIDENCE: IV. STUDY TYPE: Prognostic.

Social Media Bridges the Training Gap Between Match Day and Internship With ACGME Milestone-based Clinical Case Curriculum.

OBJECTIVES: The objective was to bridge the relative educational gap for newly matched emergency medicine preinterns between Match Day and the start of internship by implementing an Accreditation Council for Graduate Medical Education Milestone (ACGME)-based virtual case curriculum over the social media platform Slack. METHODS: We designed a Milestone-based curriculum of 10 emergency department clinical cases and used Slack to implement it. An instructor was appointed for each participating institution to lead the discussion and encourage collaboration among preinterns. Pre- and postcurriculum surveys utilized 20 statements adapted from the eight applicable Milestones to measure the evolution of preintern self-reported perceived preparedness (PP) as well as actual clinical knowledge (CK) performance on a case-based examination. RESULTS: A total of 11 institutions collaborated and 151 preinterns were contacted, 127 of whom participated. After participating in the Slack intern curriculum (SIC), preinterns reported significant improvements in PP regarding multiple Milestone topics. They also showed improved CK regarding the airway management Milestone based on examination performance. CONCLUSIONS: Implementation of our SIC may ease the difficult transition between medical school and internship for emergency medicine preinterns. Residency leadership and medical school faculty will benefit from knowledge of preintern PP, specifically of their perceived strengths and weaknesses, because this information can guide curricular focus at the end of medical school and beginning of internship. Limitations of this study include variable participation and a high attrition rate. Further studies will address the utility of such a virtual curriculum for preinterns and for rotating medical students who have been displaced from clinical rotations during the novel coronavirus pandemic.

Imaging calcium signals in vivo: a powerful tool in physiology and pharmacology.

The design and engineering of organic fluorescent Ca(2+) indicators approximately 30 years ago opened the door for imaging cellular Ca(2+) signals with a high degree of temporal and spatial resolution. Over this time, Ca(2+) imaging has revolutionized our approaches for tissue-level spatiotemporal analysis of functional organization and has matured into a powerful tool for in situ imaging of cellular activity in the living animal. In vivo Ca(2+) imaging with temporal resolution at the millisecond range and spatial resolution at micrometer range has been achieved through novel designs of Ca(2+) sensors, development of modern microscopes and powerful imaging techniques such as two-photon microscopy. Imaging Ca(2+) signals in ensembles of cells within tissue in 3D allows for analysis of integrated cellular function, which, in the case of the brain, enables recording activity patterns in local circuits. The recent development of miniaturized compact, fibre-optic-based, mechanically flexible microendoscopes capable of two-photon microscopy opens the door for imaging activity in awake, behaving animals. This development is poised to open a new chapter in physiological experiments and for pharmacological approaches in the development of novel therapies.

Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo.

Glial cell Ca2+ signals play a key role in glial-neuronal and glial-glial network communication. Numerous studies have thus far utilized cell-permeant and injected Ca2+ indicator dyes to investigate glial Ca2+ signals in vitro and in situ. Genetically encoded fluorescent Ca2+ indicators have emerged as novel probes for investigating cellular Ca2+ signals. We have expressed one such indicator protein, the YC 3.60 cameleon, under the control of the S100beta promoter and directed its expression predominantly in astrocytes and Schwann cells. Expression of YC 3.60 extended into the entire cellular cytoplasmic compartment and the fine terminal processes of protoplasmic astrocytes and Schwann cell Cajal bands. In the brain, all the cells known to express S100beta in the adult or during development, expressed YC 3.60. While expression was most extensive in astrocytes, other glial cell types that express S100beta, such as NG2 and CNP-positive oligodendrocyte progenitor cells (OP cells), microglia, and some of the large motor neurons in the brain stem, also contained YC 3.60 fluorescence. Using a variety of known in situ and in vivo assays, we found that stimuli known to elicit Ca2+ signals in astrocytes caused substantial and rapid Ca2+ signals in the YC 3.60-expressing astrocytes. In addition, forepaw stimulation while imaging astrocytes through a cranial window in the somatosensory cortex in live mice, revealed robust evoked and spontaneous Ca2+ signals. These results, for the first time, show that genetically encoded reporter is capable of recording activity-dependent Ca2+ signals in the astrocyte processes, and networks.

Synaptically released zinc triggers metabotropic signaling via a zinc-sensing receptor in the hippocampus.

Zn(2+) is coreleased with glutamate from mossy fiber terminals and can influence synaptic function. Here, we demonstrate that synaptically released Zn(2+) activates a selective postsynaptic Zn(2+)-sensing receptor (ZnR) in the CA3 region of the hippocampus. ZnR activation induced intracellular release of Ca(2+), as well as phosphorylation of extracellular-regulated kinase and Ca(2+)/calmodulin kinase II. Blockade of synaptic transmission by tetrodotoxin or CdCl inhibited the ZnR-mediated Ca(2+) rises. The responses mediated by ZnR were largely attenuated by the extracellular Zn(2+) chelator, CaEDTA, and in slices from mice lacking vesicular Zn(2+), suggesting that synaptically released Zn(2+) triggers the metabotropic activity. Knockdown of the expression of the orphan G-protein-coupled receptor 39 (GPR39) attenuated ZnR activity in a neuronal cell line. Importantly, we observed widespread GPR39 labeling in CA3 neurons, suggesting a role for this receptor in mediating ZnR signaling in the hippocampus. Our results describe a unique role for synaptic Zn(2+) acting as the physiological ligand of a metabotropic receptor and provide a novel pathway by which synaptic Zn(2+) can regulate neuronal function.

Functional reactivity of cerebral capillaries.

The spatiotemporal evolution of cerebral microcirculatory adjustments to functional brain stimulation is the fundamental determinant of the functional specificity of hemodynamically weighted neuroimaging signals. Very little data, however, exist on the functional reactivity of capillaries, the vessels most proximal to the activated neuronal population. Here, we used two-photon laser scanning microscopy, in combination with intracranial electrophysiology and intravital video microscopy, to explore the changes in cortical hemodynamics, at the level of individual capillaries, in response to steady-state forepaw stimulation in an anesthetized rodent model. Overall, the microcirculatory response to functional stimulation was characterized by a pronounced decrease in vascular transit times (20%+/-8%), a dilatation of the capillary bed (10.9%+/-1.2%), and significant increases in red blood cell speed (33.0%+/-7.7%) and flux (19.5%+/-6.2%). Capillaries dilated more than the medium-caliber vessels, indicating a decreased heterogeneity in vessel volumes and increased blood flow-carrying capacity during neuronal activation relative to baseline. Capillary dilatation accounted for an estimated approximately 18% of the total change in the focal cerebral blood volume. In support of a capacity for focal redistribution of microvascular flow and volume, significant, though less frequent, local stimulation-induced decreases in capillary volume and erythrocyte speed and flux also occurred. The present findings provide further evidence of a strong functional reactivity of cerebral capillaries and underscore the importance of changes in the capillary geometry in the hemodynamic response to neuronal activation.

Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans.

We observed a severe autosomal recessive movement disorder in mice used within our laboratory. We pursued a series of experiments to define the genetic lesion underlying this disorder and to identify a cognate disease in humans with mutation at the same locus. Through linkage and sequence analysis we show here that this disorder is caused by a homozygous in-frame 18-bp deletion in Itpr1 (Itpr1(Delta18/Delta18)), encoding inositol 1,4,5-triphosphate receptor 1. A previously reported spontaneous Itpr1 mutation in mice causes a phenotype identical to that observed here. In both models in-frame deletion within Itpr1 leads to a decrease in the normally high level of Itpr1 expression in cerebellar Purkinje cells. Spinocerebellar ataxia 15 (SCA15), a human autosomal dominant disorder, maps to the genomic region containing ITPR1; however, to date no causal mutations had been identified. Because ataxia is a prominent feature in Itpr1 mutant mice, we performed a series of experiments to test the hypothesis that mutation at ITPR1 may be the cause of SCA15. We show here that heterozygous deletion of the 5' part of the ITPR1 gene, encompassing exons 1-10, 1-40, and 1-44 in three studied families, underlies SCA15 in humans.

Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store.

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.

Signaling proteins in raft-like microdomains are essential for Ca2+ wave propagation in glial cells.

The hypothesis that calcium signaling proteins segregate into lipid raft-like microdomains was tested in isolated membranes of rat oligodendrocyte progenitor (OP) cells and astrocytes using Triton X-100 solubilization and density gradient centrifugation. Western blot analysis of gradient fractions showed co-localization of caveolin-1 with proteins involved in the Ca2+ signaling cascade. These included agonist receptors, P2Y1, and M1, TRPC1, IP3R2, ryanodine receptor, as well as the G protein Galphaq and Homer. Membranes isolated from agonist-stimulated astrocytes showed an enhanced recruitment of phospholipase C (PLCbeta1), IP3R2 and protein kinase C (PKC-alpha) into lipid raft fractions. IP3R2, TRPC1 and Homer co-immunoprecipitated, suggesting protein-protein interactions. Disruption of rafts by cholesterol depletion using methyl-beta-cyclodextrin (beta-MCD) altered the distribution of caveolin-1 and GM1 to non-raft fractions with higher densities. beta-MCD-induced disruption of rafts inhibited agonist-evoked Ca2+ wave propagation in astrocytes and attenuated wave speeds. These results indicate that in glial cells, Ca2+ signaling proteins might exist in organized membrane microdomains, and these complexes may include proteins from different cellular membrane systems. Such an organization is essential for Ca2+ wave propagation.

Signaling proteins in the axoglial apparatus of sciatic nerve nodes of Ranvier.

During action potential conduction, the axonal specializations at the node, together with the adjacent paranodal terminations of the myelin sheath, interact with glial processes that invest the nodal gap. The nature of the mutual signals between axons and myelinating glia, however, are not well understood. Here we have characterized the distribution of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in the axoglial apparatus by immunohistochemistry, using known myelin domain-specific markers. While IP(3)R1 is not expressed in the Schwann cells or the axon, IP(3)R2 and IP(3)R3 are expressed in distinct cellular domains, suggesting distinct signaling roles for the two receptors. IP(3)R3 is the most predominant isoform in Schwann cells, and is expressed in particularly dense patches in the paranodal region. In addition to IP(3)Rs, two other members of the metabotropic Ca(2+) signaling pathway, G(alpha)q, and P(2)Y1 type of purinoceptors were also found in Schwann cells. Their pattern of expression matches the expression of their signaling partners, the IP(3)Rs. One interesting finding to emerge from this study is the expression of connexin 32 (Cx32) in close proximity with IP(3)R3. Although IP(3)R3 and Cx32 are not colocalized, their expression in the same membrane areas raises the question whether Schwann cell Ca(2+) signals either control the function of the gap junctions, or whether the gap junctional channels serve as conduits for rapid radial spread of Ca(2+) signals initiated during action potential propagation.

An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva.

Barnacles, like many marine invertebrates, cause serious biofouling to marine industrial constructions and hulls of vessels as they attach themselves to such surfaces. Precise biochemical understanding of the underwater adhesion to surfaces requires a detailed characterization of the biology of the control of barnacle cement secretion and the proteins that make up the cement. In this study, we have investigated cement secretion by cyprid larvae of Balanus improvisus (D.) and the morphology of their cement glands. We studied the cement protein organization within cement granules and categorized the granules into four different types according to their size and morphology, before and after stimulation of secretion. In addition, we followed the exocytotic process of cement secretion in vivo and discovered that granules undergo a dramatic swelling during secretion. Such swelling might be due to an increased osmotic activity of granule contents, following a process of hydration. We hypothesize that this hydration is essential for exocytotic secretion and conclude that cement protein exocytosis is a more complex process than previously thought and is similar to exocytotic secretion in vertebrate systems, such as histamine secretion from mast cells and exocrine secretion in the salivary gland and the pancreas.

Metabotropic glutamate receptors and dopamine receptors cooperate to enhance extracellular signal-regulated kinase phosphorylation in striatal neurons.

Striatal medium spiny neurons are an important site of convergence for signaling mediated by the neurotransmitters dopamine and glutamate. We report that in striatal neurons in primary culture, signaling through group I metabotropic glutamate receptors (mGluRs) 1/5 and the D1 class of dopamine receptors (DRs) 1/5 converges to increase phosphorylation of the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2). Induction of mitogen-activated protein kinase kinase-dependent signaling cascades by either mGluR1/5 or DR1/5 gave rise to increases in phosphorylation of ERK2. Coactivation of mGluR1/5 and DR1/5 with (S)-3,5-dihydroxyphenylglycine and (+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride enhanced the phosphorylation of ERK2. This interaction between mGluR1/5 and DR1/5 required protein kinase C (PKC), because the PKC inhibitors calphostin C, bisindolylmaleimide I, and Gö6976 blocked DR1/5-enhanced phosphorylation of ERK2. Use of the phosphatase inhibitors calyculin and okadaic acid indicated that inhibition of protein phosphatases 1 and 2A dramatically enhanced ERK2 phosphorylation by mGluR1/5. Coactivation of mGluR1/5 and DR1/5 also enhanced cAMP-response element binding protein (CREB) phosphorylation (compared with each receptor agonist alone) but did not enhance CREB-mediated transcriptional activity. Thus, signal transduction pathways activated by DR1/5 and mGluR5 interact to modify downstream events in striatal neurons while retaining numerous regulatory checkpoints.

Vanilloid receptor 1 regulates multiple calcium compartments and contributes to Ca2+-induced Ca2+ release in sensory neurons.

Vanilloid receptor 1 belongs to the transient receptor potential ion channel family and transduces sensations of noxious heat and inflammatory hyperalgesia in nociceptive neurons. These neurons contain two vanilloid receptor pools, one in the plasma membrane and the other in the endoplasmic reticulum. The present experiments characterize these two pools and their functional significance using calcium imaging and 45Ca uptake in stably transfected cells or dorsal root ganglion neurons. The plasma membrane localized receptor is directly activated by vanilloids. The endoplasmic reticulum pool was demonstrated to be independently activated with 20 microm capsaicin or 1.6 microm resiniferatoxin using a bathing solution containing 10 microm Ruthenium Red (to selectively block plasma membrane-localized receptors) and 100 microm EGTA. We also demonstrate an overlap between the endoplasmic reticulum-localized vanilloid receptor regulated stores and thapsigargin-sensitive stores. Direct depletion of calcium via activation of endoplasmic reticulum-localized vanilloid receptor 1 triggered store operated calcium entry. Furthermore, we found that, in the presence of low extracellular calcium (10(-5) m), either 2 microm capsaicin or 0.1 nm-1.6 microm resiniferatoxin caused a pronounced calcium-induced calcium release in either vanilloid receptor-expressing neurons or heterologous expression systems. This phenomenon may allow new insight into how nociceptive neuron function in response to a variety of nociceptive stimuli both acutely and during prolonged nociceptive signaling.

Astrocytes in adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors.

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.

Mitochondria regulate Ca2+ wave initiation and inositol trisphosphate signal transduction in oligodendrocyte progenitors.

Mitochondria in oligodendrocyte progenitor cells (OPs) take up and release cytosolic Ca2+ during agonist-evoked Ca2+ waves, but it is not clear whether or how they regulate Ca2+ signaling in OPs. We asked whether mitochondria play an active role during agonist-evoked Ca2+ release from intracellular stores. Ca2+ puffs, wave initiation, and wave propagation were measured in fluo-4 loaded OP processes using linescan confocal microscopy. Mitochondrial depolarization, measured by tetramethyl rhodamine ethyl ester (TMRE) fluorescence, accompanied Ca2+ puffs and waves. In addition, waves initiated only where mitochondria were localized. To determine whether energized mitochondria were necessary for wave generation, we blocked mitochondrial function with the electron transport chain inhibitor antimycin A (AA) in combination with oligomycin. AA decreased wave speed and puff probability. These effects were not due to global changes in ATP. We found that AA increased cytosolic Ca2+, markedly reduced agonist-evoked inositol trisphosphate (IP3) production, and also enhanced phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the Ca2+ dependent protein gelsolin. Thus, the reduction in puff probability and wave speed after AA treatment may be explained by competition for PIP2 between phospholipase C and gelsolin. Energized mitochondria and low cytosolic Ca2+ concentration may be required to maintain PIP2, a substrate for IP3 signal transduction.