Nicotinamide mononucleotide adenylyltransferase promotes hypoxic survival by activating the mitochondrial unfolded protein response.

Gain-of-function mutations in the mouse nicotinamide mononucleotide adenylyltransferase type 1 (Nmnat1) produce two remarkable phenotypes: protection against traumatic axonal degeneration and reduced hypoxic brain injury. Despite intensive efforts, the mechanism of Nmnat1 cytoprotection remains elusive. To develop a new model to define this mechanism, we heterologously expressed a mouse Nmnat1 non-nuclear-localized gain-of-function mutant gene (m-nonN-Nmnat1) in the nematode Caenorhabditis elegans and show that it provides protection from both hypoxia-induced animal death and taxol-induced axonal pathology. Additionally, we find that m-nonN-Nmnat1 significantly lengthens C. elegans lifespan. Using the hypoxia-protective phenotype in C. elegans, we performed a candidate screen for genetic suppressors of m-nonN-Nmnat1 cytoprotection. Loss of function in two genes, haf-1 and dve-1, encoding mitochondrial unfolded protein response (mitoUPR) factors were identified as suppressors. M-nonN-Nmnat1 induced a transcriptional reporter of the mitoUPR gene hsp-6 and provided protection from the mitochondrial proteostasis toxin ethidium bromide. M-nonN-Nmnat1 was also protective against axonal degeneration in C. elegans induced by the chemotherapy drug taxol. Taxol markedly reduced basal expression of a mitoUPR reporter; the expression was restored by m-nonN-Nmnat1. Taken together, these data implicate the mitoUPR as a mechanism whereby Nmnat1 protects from hypoxic and axonal injury.

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans.

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2-3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.

Nitrous oxide (N(2)O) requires the N-methyl-D-aspartate receptor for its action in Caenorhabditis elegans.

Nitrous oxide (N(2)O, also known as laughing gas) and volatile anesthetics (VAs), the original and still most widely used general anesthetics, produce anesthesia by ill-defined mechanisms. Electrophysiological experiments in vertebrate neurons have suggested that N(2)O and VAs may act by distinct mechanisms; N(2)O antagonizes the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors, whereas VAs alter the function of a variety of other synaptic proteins. However, no genetic or pharmacological experiments have demonstrated that any of these in vitro actions are responsible for the behavioral effects of either class of anesthetics. By using genetic tools in Caenorhabditis elegans, we tested whether the action of N(2)O requires the NMDA receptor in vivo and whether its mechanism is shared by VAs. Distinct from the action of VAs, N(2)O produced behavioral defects highly specific and characteristic of that produced by loss-of-function mutations in both NMDA and non-NMDA glutamate receptors. A null mutant of nmr-1, which encodes a C. elegans NMDA receptor, was completely resistant to the behavioral effects of N(2)O, whereas a non-NMDA receptor-null mutant was normally sensitive. The N(2)O-resistant nmr-1(null) mutant was not resistant to VAs. Likewise, VA-resistant mutants had wild-type sensitivity to N(2)O. Thus, the behavioral effects of N(2)O require the NMDA receptor NMR-1, consistent with the hypothesis formed from vertebrate electrophysiological data that a major target of N(2)O is the NMDA receptor.

The effect of prone positioning on intraocular pressure in anesthetized patients.

BACKGROUND: Ocular perfusion pressure is commonly defined as mean arterial pressure minus intraocular pressure (IOP). Changes in mean arterial pressure or IOP can affect ocular perfusion pressure. IOP has not been studied in this context in the prone anesthetized patient. METHODS: After institutional human studies committee approval and informed consent, 20 patients (American Society of Anesthesiologists physical status I-III) without eye disease who were scheduled for spine surgery in the prone position were enrolled. IOP was measured with a Tono-pen XL handheld tonometer at five time points: awake supine (baseline), anesthetized (supine 1), anesthetized prone (prone 1), anesthetized prone at conclusion of case (prone 2), and anesthetized supine before wake-up (supine 2). Anesthetic protocol was standardized. The head was positioned with a pinned head-holder. Data were analyzed with repeated-measures analysis of variance and paired t test. RESULTS: Supine 1 IOP (13 +/- 1 mmHg) decreased from baseline (19 +/- 1 mmHg) (P < 0.05). Prone 1 IOP (27 +/- 2 mmHg) increased in comparison with baseline (P < 0.05) and supine 1 (P < 0.05). Prone 2 IOP (40 +/- 2 mmHg) was measured after 320 +/- 107 min in the prone position and was significantly increased in comparison with all previous measurements (P < 0.05). Supine 2 IOP (31 +/- 2 mmHg) decreased in comparison with prone 2 IOP (P < 0.05) but was relatively elevated in comparison with supine 1 and baseline (P < 0.05). Hemodynamic and ventilatory parameters remained unchanged during the prone period. CONCLUSIONS: Prone positioning increases IOP during anesthesia. Ocular perfusion pressure could therefore decrease, despite maintenance of normotension.

Enantiospecificity of cholesterol function in vivo.

The importance of the absolute configuration of cholesterol for its function in vivo is unknown. To directly test this question in vivo, we synthesized the enantiomer of cholesterol (ent-cholesterol) and tested its ability to substitute for natural cholesterol (nat-cholesterol) in the growth, viability, and behavior of Caenorhabditis elegans, a cholesterol auxotroph. First-generation animals grown on ent-cholesterol were viable with only mild behavioral defects. However, ent-cholesterol produced 100% lethality/arrest of their second generation progeny. Isotopically labeled ent-cholesterol incorporated into animals, indicating that its lethality was not secondary to cholesterol starvation. When mixed with nat-cholesterol, ent-cholesterol was not inert; rather, it antagonized the activity of nat-cholesterol. These results demonstrate for the first time that the absolute configuration of cholesterol, not just its physical properties, is essential for its functions in vivo.

Goalpha regulates volatile anesthetic action in Caenorhabditis elegans.

To identify genes controlling volatile anesthetic (VA) action, we have screened through existing Caenorhabditis elegans mutants and found that strains with a reduction in Go signaling are VA resistant. Loss-of-function mutants of the gene goa-1, which codes for the alpha-subunit of Go, have EC(50)s for the VA isoflurane of 1.7- to 2.4-fold that of wild type. Strains overexpressing egl-10, which codes for an RGS protein negatively regulating goa-1, are also isoflurane resistant. However, sensitivity to halothane, a structurally distinct VA, is differentially affected by Go pathway mutants. The RGS overexpressing strains, a goa-1 missense mutant found to carry a novel mutation near the GTP-binding domain, and eat-16(rf) mutants, which suppress goa-1(gf) mutations, are all halothane resistant; goa-1(null) mutants have wild-type sensitivities. Double mutant strains carrying mutations in both goa-1 and unc-64, which codes for a neuronal syntaxin previously found to regulate VA sensitivity, show that the syntaxin mutant phenotypes depend in part on goa-1 expression. Pharmacological assays using the cholinesterase inhibitor aldicarb suggest that VAs and GOA-1 similarly downregulate cholinergic neurotransmitter release in C. elegans. Thus, the mechanism of action of VAs in C. elegans is regulated by Goalpha, and presynaptic Goalpha-effectors are candidate VA molecular targets.

The effects of clonidine premedication and scalp infiltration of lidocaine on hemodynamic responses to laryngoscopy and skull pin head-holder insertion during skull base procedures.

This study was designed to determine if oral clonidine or lidocaine, injected into the scalp before head-holder (H-H) insertion, would attenuate the hemodynamic effects associated with intubation and H-H placement. Thirty-four patients undergoing skull base procedures were randomized to four groups. Group 1 received clonidine 5 mcg/kg po before surgery with 10 to 15 ml of 1% lidocaine infiltrated at pin insertion sites; Group 2 received clonidine with saline infiltration; Group 3 received a placebo preoperatively and had lidocaine infiltrated at pin sites; and Group 4 received a placebo with saline infiltrated. All patients had a standard anesthetic titrated to a 10 to 14 Hz EEG endpoint during laryngoscopy and H-H placement. Mean arterial pressure (MAP) was similar between groups during intubation, but heart rate (HR) increased in patients who did not receive clonidine. H-H application increased HR and MAP in Group 4. HR also increased after H-H placement in patients who received oral clonidine, while patients receiving scalp lidocaine or both clonidine and scalp lidocaine had little change in either value. Clonidine attenuated HR increases after laryngoscopy but not after H-H placement. Lidocaine injected at the pin sites reduced HR, and MAP increased after H-H insertion. The combination of oral clonidine and scalp lidocaine blunted hemodynamic responses to both intubation and H-H placement.

A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans.

The molecular mechanisms underlying general anesthesia are unknown. For volatile general anesthetics (VAs), indirect evidence for both lipid and protein targets has been found. However, no in vivo data have implicated clearly any particular lipid or protein in the control of sensitivity to clinical concentrations of VAs. Genetics provides one approach toward identifying these mechanisms, but genes strongly regulating sensitivity to clinical concentrations of VAs have not been identified. By screening existing mutants of the nematode Caenorhabditis elegans, we found that a mutation in the neuronal syntaxin gene dominantly conferred resistance to the VAs isoflurane and halothane. By contrast, other mutations in syntaxin and in the syntaxin-binding proteins synaptobrevin and SNAP-25 produced VA hypersensitivity. The syntaxin allelic variation was striking, particularly for isoflurane, where a 33-fold range of sensitivities was seen. Both the resistant and hypersensitive mutations decrease synaptic transmission; thus, the indirect effect of reducing neurotransmission does not explain the VA resistance. As assessed by pharmacological criteria, halothane and isoflurane themselves reduced cholinergic transmission, and the presynaptic anesthetic effect was blocked by the resistant syntaxin mutation. A single gene mutation conferring high-level resistance to VAs is inconsistent with nonspecific membrane-perturbation theories of anesthesia. The genetic and pharmacological data suggest that the resistant syntaxin mutant directly blocks VA binding to or efficacy against presynaptic targets that mediate anesthetic behavioral effects. Syntaxin and syntaxin-binding proteins are candidate anesthetic targets.

A quantitative genetic approach towards volatile anesthetic mechanisms in C. elegans.

Quantitative genetics is the study of the heritability of continuous traits such as height or IQ. Quantitative trait loci (QTLs) represent the genes that are responsible for these quantitative traits. Sensitivity to the volatile anesthetic halothane is a genetically controlled quantitative trait in the nematode C. elegans. The QTLs that are responsible for the 12-fold range in halothane EC50 in these strains map to a few places with at least one major effect locus on chromosome V. Congenic strains for chromosome V confirmed these loci and offer the means to finely map them for positional cloning.

Convection versus conduction cooling for induction of mild hypothermia during neurovascular procedures in adults.

Hypothermia for cerebral protection is usually achieved by administration of intravenous fluids at room temperature, cooling ambient air, ice packs, and a temperature-adjustable circulating water mattress. We compared cooling by conduction by using a water mattress to cool by convection by using a forced-air cooling device. Twenty patients were prospectively randomized to two groups: 10 patients cooled by convection (CC) and 10 patients cooled by traditional methods (TC). Two patients in the CC group were withdrawn from the study and excluded from the analysis; one patient for failure to cool despite the use of both techniques, and the other for the abrupt onset of arrhythmias and myocardial depression during hypothermia. Temperature was measured at the tympanic membrane, pulmonary artery, and esophageal probe sites and recorded every 15 min. The time required to reach the target temperature range of 33-34 degrees C was recorded. We found no differences in the temperatures measured at the three sites during cooling and rewarming. Baseline temperatures recorded from the pulmonary artery catheter before beginning "active cooling" were similar in both groups (TC, 35.0 +/- 0.2 degrees C vs. CC, 35.3 +/- 0.1 degrees C). We found no difference in the time to target temperature between TC and CC (TC, 178 +/- 25 min vs. CC, 142 +/- 21 min). One patient had some arrhythmias on cooling in the convective group, but her preoperative condition may have been responsible. In conclusion, cooling by convection appears to be a safe alternative to conduction cooling.

Quantitative trait loci controlling halothane sensitivity in Caenorhabditis elegans.

Genetic analysis is an essential tool for defining the molecular mechanisms whereby volatile anesthetics (VA) disrupt nervous system function. However, the degree of natural variation of the genetic determinants of VA sensitivity has not been determined nor have mutagenesis approaches been very successful at isolating significantly resistant mutant strains. Thus, a quantitative genetic approach was taken toward these goals. Recombinant-inbred strains derived from two evolutionarily distinct lineages of the nematode Caenorhabditis elegans were tested for sensitivity to clinically relevant concentrations (0.3-0.5 mM) of the VA halothane. The halothane sensitivities of coordinated movement and male mating behavior were highly variant among the recombinant-inbred strains with a range of EC50 values of 13- and 4-fold, respectively. Both traits were highly heritable (H2 = 0.82, 0.87, respectively). Several strains were found to be significantly resistant to halothane when compared with the wild-type strain N2. A major locus or loci mapping to the middle of chromosome V accounted for more than 40% of the phenotypic variance for both traits. Five weaker loci, four of which interact, explained most of the remaining variance. None of the halothane-sensitivity quantitative trait loci significantly affected behavior in the absence of halothane or halothane's potency for C. elegans immobilization, which requires 5-fold higher drug concentrations. Thus, the quantitative trait loci are unlikely to result from differences in halothane-independent (native) behavior or differences in halothane metabolism or permeability. Rather, these loci may code for targets and/or downstream effectors of halothane in the C. elegans nervous system or for modifiers of such gene products.

Behavioral effects of volatile anesthetics in Caenorhabditis elegans.

BACKGROUND: The nematode Caenorhabditis elegans offers many advantages as a model organism for studying volatile anesthetic actions. It has a simple, well-understood nervous system; it allows the researcher to do forward genetics; and its genome will soon be completely sequenced. C. elegans is immobilized by volatile anesthetics only at high concentrations and with an unusually slow time course. Here other behavioral dysfunctions are considered as anesthetic endpoints in C. elegans. METHODS: The potency of halothane for disrupting eight different behaviors was determined by logistic regression of concentration and response data. Other volatile anesthetics were also tested for some behaviors. Established protocols were used for behavioral endpoints that, except for pharyngeal pumping, were set as complete disruption of the behavior. Time courses were measured for rapid behaviors. Recovery from exposure to 1 or 4 vol% halothane was determined for mating, chemotaxis, and gross movement. All experiments were performed at 20 to 22 degrees C. RESULTS: The median effective concentration values for halothane inhibition of mating (0.30 vol%-0.21 mM), chemotaxis (0.34 vol%-0.24 mM), and coordinated movement (0.32 vol% - 0.23 mM) were similar to the human minimum alveolar concentration (MAC; 0.21 mM). In contrast, halothane produced immobility with a median effective concentration of 3.65 vol% (2.6 mM). Other behaviors had intermediate sensitivities. Halothane's effects reached steady-state in 10 min for all behaviors tested except immobility, which required 2 h. Recovery was complete after exposure to 1 vol% halothane but was significantly reduced after exposure to immobilizing concentrations. CONCLUSIONS: Volatile anesthetics selectively disrupt C. elegans behavior. The potency, time course, and recovery characteristics of halothane's effects on three behaviors are similar to its anesthetic properties in vertebrates. The affected nervous system molecules may express structural motifs similar to those on vertebrate anesthetic targets.

Jugular bulb temperature: comparison with brain surface and core temperatures in neurosurgical patients during mild hypothermia.

Blood temperature at the jugular bulb was monitored in 10 patients undergoing neurovascular procedures that used induced mild hypothermia, and its correlation with surface brain, core, and peripheral temperatures was determined. The study was motivated by the difficulty encountered in directly measuring global brain temperature and the poor correlations between various core and peripheral sites temperatures and brain temperature, particularly during deep hypothermia. Although not statistically significant, previous studies have suggested a trend toward higher brain temperatures. Temperatures from the jugular bulb (collected using a No. 5 French Swan-Ganz catheter) as well as from subdural, pulmonary artery, esophagus, tympanic membrane, and bladder sites were analyzed during three surgical conditions: prior to incision, with the dura open, and after closure of the dura. No complications related to placement of the jugular bulb catheter, induced hypothermia, or temperature monitoring were seen. The authors found that jugular bulb temperature was similar to pulmonary artery and esophageal temperatures; although prior to incision it tended to be higher than that found at the pulmonary artery, most commonly by 0.2 degrees C. Surface brain temperature was cooler than all other temperatures (p < 0.05), except that of the tympanic membrane, and was particularly sensitive to environmental variations. Finally, as has been shown by others, bladder temperature lagged substantially behind core temperatures particularly during rapid cooling and rewarming of the patient. In summary, monitoring of jugular bulb temperature is a feasible technique, and temperatures measured in the jugular bulb are similar to core temperatures.

Stepwise activation of the mouse acetylcholine receptor delta- and gamma-subunit genes in clonal cell lines.

We used the DNase I-hypersensitive sites around the mouse acetylcholine receptor delta-subunit gene as a guide toward the cloning and sequencing of delta and gamma transcriptional regulatory regions and as a means to assess chromatin structural activation of the delta- and gamma-subunit genes during myogenesis. Genomic cloning of hypersensitive sites downstream of the delta-subunit gene revealed the presence of the gamma-subunit gene approximately 5 kilobases away; the hypersensitive sites mapped to the 5' end of the gamma-subunit gene. Sequence comparison of restriction fragments containing hypersensitive sites in analogous locations at the 5' ends of the delta- and gamma-subunit genes uncovered little overall homology between the two genomic fragments; however, an 11- of 13-base-pair match between the two sequences was found. Homologs to this sequence were also found to be present in the upstream regions of the chicken alpha- and mouse beta-subunit genes. By RNase protection and primer extension analyses, the delta-subunit gene transcription start site was mapped to 56 base pairs upstream of the initiator ATG codon. Clonal cell lines with various potentials to differentiate to the skeletal muscle phenotype were examined for their hypersensitive site pattern within the delta-gamma locus. Only remote hypersensitive sites flanking the locus appear in pluripotential mesodermal cells. A cell line of determined but inducible myoblasts expressed only one more intergenic site, while in permissively differentiating myoblasts hypersensitive sites were already present at the 5' ends of the delta and gamma genes prior to differentiation. Terminal differentiation resulted in an identical pattern of hypersensitive sites in all muscle cell lines examined so far, with an intergenic site near the gamma-subunit gene being the only site specific to the differentiated muscle phenotype.

Studies of acetylcholine receptor subunit gene expression: chromatin structural changes during myogenesis.

Myogenesis proceeds stepwise from pluripotential stem cell to differentiated myotube. The precise number of transitions that occur along the developmental pathway remains to be determined. We examined the myogenic pathway as modelled by mouse mesodermal stem cell and muscle cell lines for stage-specific alterations in the chromatin structure of the acetylcholine receptor delta and gamma subunit genes. We reasoned that such an analysis would allow us to observe either the primary events in the activation of these muscle-specific genes or processes secondary to the binding of muscle-specific regulatory proteins. We probed chromatin structure with DNase I (deoxyribonuclease I) and precisely mapped to the 5' ends of the delta and gamma genes DNase I hypersensitive (DH) sites whose induction is unique to each myogenic stage. Putative mesodermal stem cells have the simplest pattern of DH sites with no sites near the 5' ends of the delta and gamma genes, whereas differentiated myotubes express the most complex pattern; the myoblast pattern is intermediate and of two types. In muscle cell lines where differentiation must be induced the myoblasts have a simple pattern (one more site than stem cells); in muscle lines where differentiation is spontaneous the myoblasts express a complex pattern of DH sites (one less site than myotubes). Inducible myoblasts seem to be arrested in an earlier step in the myogenic pathway than spontaneously differentiating myoblasts. Thus, myogenic activation of acetylcholine receptor subunit genes appears to be a stepwise process that can be detected by chromatin structural changes specific to four distinct stages of muscle development: stem cell, early myoblast, late myoblast, and differentiated myotube.

DNase I-hypersensitive sites surround the mouse acetylcholine receptor delta-subunit gene.

The mouse skeletal muscle acetylcholine receptor is regulated, at least in part, by transcriptional mechanisms. As a first step toward defining the cis- and trans-acting factors involved in acetylcholine receptor transcriptional regulation, we examined mouse muscle and fibroblast cells for the presence of DNase I-hypersensitive sites in the chromatin surrounding the delta-subunit gene. We detect six DNase I-hypersensitive sites in muscle DNA blots hybridized with a mouse delta-subunit cDNA. Two hypersensitive sites lie distantly 5' to the delta gene, one lies near the 5' end of the delta gene, one lies near its 3' end, and two hypersensitive sites lie distantly 3'. The pair of distantly 3' hypersensitive sites should by analogy to chicken and human genomes lie near the gamma-subunit gene. Only the distantly 5' pair of hypersensitive sites is present in fibroblasts; the remainder is muscle specific. Two hypersensitive sites, one at the 3' end of the delta gene and one distantly 3', appear only after terminal muscle differentiation and, therefore, are developmentally regulated, similarly to receptor subunit mRNAs. All four muscle-specific hypersensitive sites are excellent candidates for regulatory loci for the delta- and gamma-subunit genes of mouse acetylcholine receptor.