HIV Infection Is Associated with Loss of Anti-Inflammatory Alveolar Macrophages.

HIV type 1 is associated with pulmonary dysfunction that is exacerbated by cigarette smoke. Alveolar macrophages (AM) are the most prominent immune cell in the alveolar space. These cells play an important role in clearing inhaled pathogens and regulating the inflammatory environment; however, how HIV infection impacts AM phenotype and function is not well understood, in part because of their autofluorescence and the absence of well-defined surface markers. The main aim of this study was to evaluate the impact of HIV infection on human AM and to compare the effect of smoking on their phenotype and function. Time-of-flight mass cytometry and RNA sequencing were used to characterize macrophages from human bronchoalveolar lavage of HIV-infected and -uninfected smokers and nonsmokers. We found that the frequency of CD163+ anti-inflammatory AM was decreased, whereas CD163-CCR7+ proinflammatory AM were increased in HIV infection. HIV-mediated proinflammatory polarization was associated with increased levels of inflammatory cytokines and macrophage activation. Conversely, smoking heightened the inflammatory response evident by change in the expression of CXCR4 and TLR4. Altogether, these findings suggest that HIV infection, along with cigarette smoke, favors a proinflammatory macrophage phenotype associated with enhanced expression of inflammatory molecules. Further, this study highlights time-of-flight mass cytometry as a reliable method for immunophenotyping the highly autofluorescent cells present in the bronchoalveolar lavage of cigarette smokers.

Upregulation of Chitinase 1 in Alveolar Macrophages of HIV-Infected Smokers.

Recent studies suggest that HIV infection is an independent risk factor for the development of chronic obstructive pulmonary disease (COPD). We hypothesized that HIV infection and cigarette smoking synergize to alter the function of alveolar macrophages (AMs). To test this hypothesis, global transcriptome analysis was performed on purified AMs from 20 individuals split evenly between HIV-uninfected nonsmokers and smokers and untreated HIV-infected nonsmokers and smokers. Differential expression analysis identified 143 genes significantly altered by the combination of HIV infection and smoking. Of the differentially expressed genes, chitinase 1 (CHIT1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1), both previously associated with COPD, were among the most upregulated genes (5- and 26-fold, respectively) in the untreated HIV-infected smoker cohort compared with HIV-uninfected nonsmokers. Expression of CHIT1 and CYP1B1 correlated with the expression of genes involved in extracellular matrix organization, oxidative stress, immune response, and cell death. Using time-of-flight mass cytometry to characterize AMs, a significantly decreased expression of CD163, an M2 marker, was seen in HIV-infected subjects, and CD163 inversely correlated with CYP1B1 expression in AMs. CHIT1 protein levels were significantly upregulated in bronchoalveolar lavage fluid from HIV-infected smokers, and increased CHIT1 levels negatively correlated with lung function measurements. Overall, these findings raise the possibility that elevated CHIT1 and CYP1B1 are early indicators of COPD development in HIV-infected smokers that may serve as biomarkers for determining this risk.

Degradation of SAMHD1 by Vpx Is Independent of Uncoating.

UNLABELLED: Sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm. IMPORTANCE: SAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.

Chest compressions do not disrupt the seal created by the laryngeal mask airway during positive pressure ventilation: a preliminary porcine study.

OBJECTIVE: Pulmonary aspiration of gastric contents occurs 20 to 30% of the time during cardiopulmonary resuscitation (CPR) of cardiac arrest due to loss of protective airway reflexes, pressure changes generated during CPR, and positive pressure ventilation (PPV). Although the American Heart Association has recommended the laryngeal mask airway (LMA) as an acceptable alternative airway for use by emergency medical service personnel, concerns over the capacity of the device to protect from pulmonary aspiration remain. We sought to determine the occurrence of aspiration after LMA placement, CPR, and PPV. METHODS: We inserted a size 4 LMA, modified so that a vacuum catheter could be advanced past the LMA diaphragm, into the hypopharynx of 16 consecutive postexperimental mixed-breed domestic swine. Fifteen millilitres of heparinized blood was instilled into the oropharynx. Chest compressions were performed for 60 seconds with asynchronous ventilation via a mechanical ventilator. We then suctioned through the LMA for 1 minute. The catheter was removed and inspected for signs of blood. The LMA cuff was deflated, removed, and inspected for signs of blood. RESULTS: None of 16 animals (95% CI 0-17%) had a positive test for the presence of blood in both the vacuum catheter and the intima of the LMA diaphragm. CONCLUSIONS: In this swine model of regurgitation after LMA placement, there were no cases with evidence of blood beyond the seal created by the LMA cuff. Future studies are needed to determine the frequency of pulmonary aspiration after LMA placement during CPR and PPV in the clinical setting.

A DNA sequence recognition loop on APOBEC3A controls substrate specificity.

APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.

The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus.

Sterile alpha motif domain- and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.

SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.

SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.

Quantitative waveform measures of the electrocardiogram as continuous physiologic feedback during resuscitation with cardiopulmonary bypass.

BACKGROUND: There are few if any real-time physiologic measures that currently provide feedback during resuscitation from cardiac arrest. Such measures could be used to guide therapy not simply based on process guidelines but on the physiologic response of the patient from moment to moment. To this end, we applied an existing technology - quantitative waveform measures (QWMs) of the ventricular fibrillation (VF) electrocardiogram (ECG) - as a continuous measure of myocardial response to reperfusion with cardiopulmonary bypass (CPB) after prolonged cardiac arrest. METHODS: Sixteen domestic, mixed-breed swine were sedated, anesthetized and paralyzed. Mechanical ventilation with room air was provided. Large diameter bypass catheters were placed in the right external jugular vein and right femoral artery for cardiopulmonary bypass (CPB). VF was induced with a 3-s 100mA transthoracic shock and left untreated for 15, 20, 25, or 30min, followed by 10min of centrifugal pump CPB (Bard CPS). Continuous Lead II ECG was recorded with an electronic data acquisition system (Power Lab, ADInstruments). Four QWMs representing 4 signal characteristics of the VF ECG were calculated in 5-s windows throughout the course of untreated VF and resuscitation with CPB. RESULTS: Four animals were assigned to each VF duration group. QWM recovery was inversely correlated with untreated VF duration, and was drastically reduced above 20min of untreated VF. Return of spontaneous circulation (ROSC) was highly unlikely after 20min of untreated VF. CONCLUSION: QWMs of the VF ECG provided a real-time metric of myocardial electrophysiologic response to reperfusion with CPB. Resuscitation from greater than 20min of untreated cardiac arrest was unlikely. QWMs may be useful for titrating CPB duration before defibrillation and assessing CPR quality independently of process guidelines.

Feasibility of initiating extracorporeal life support during mechanical chest compression CPR: a porcine pilot study.

BACKGROUND: Recently, portable extracorporeal membrane oxygenation (ECMO) machines have become commercially available. This creates the potential to utilize extracorporeal life support (ECLS) for the treatment of sudden cardiac arrest in the emergency department, and potentially in the out-of-hospital setting. OBJECTIVE: We sought to determine the feasibility of installing the ECMO circuit during delivery of mechanical chest compression CPR. METHODS: We used 5 mixed-breed domestic swine with a mean mass of 26.0 kg. After induction of anesthesia, animals were instrumented with micromanometer-tipped transducers placed in the aorta and right atrium via the left femoral artery and vein. Ventricular fibrillation (VF) was induced electrically with a transthoracic shock and left untreated for 8 min. Then, mechanical chest compressions were begun (LUCAS, Jolife, Lund, Sweden) and manual ventilations were performed to maintain ETCO(2) between 35 and 45Torr. Compressions continued until ECMO flow was started. Ten minutes after induction of VF, drugs were given (epinephrine, vasopressin, and propranolol). ECMO installation was started via cutdown on the right external jugular vein and right femoral artery for placement of venous and arterial catheters while chest compressions continued. ECMO installation start time varied from 17 to 30 min after start of compressions and continued until ECG indicated a shockable rhythm. First rescue shocks were given at 22, 32, 35, 44, and 65 min. RESULTS: ECMO was successfully installed in all five animals without incident. It was necessary to briefly discontinue chest compressions during the most delicate part of inserting the catheters into the vessels. ECMO also allowed for very rapid cooling of the animals and facilitated post-resuscitation hemodynamic support. Only the 65-min animal did not attain return of spontaneous circulation (ROSC). CONCLUSION: Mechanical chest compression may be a suitable therapeutic bridge to the installation of ECMO and does not interfere with ECMO catheter placement.

The cargo-binding domain of transportin 3 is required for lentivirus nuclear import.

Lentiviruses, unlike the gammaretroviruses, are able to infect nondividing cells by transiting through nuclear pores to access the host genomic DNA. Several nuclear import and nuclear pore components have been implicated as playing a role in nuclear import, including transportin 3 (TNPO3), a member of the importin-ß family of nuclear import proteins. We demonstrated that TNPO3 was required by several lentiviruses, with simian immunodeficiency virus mac239 (SIVmac239) and equine infectious anemia virus (EIAV) the most dependent and human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) the least. Analysis of HIV-1/SIVmac239 chimeric viruses showed that dependence on TNPO3 mapped to the SIVmac239 capsid. Mutation of a single amino acid, A76V in the SIVmac239 capsid, rendered the virus TNPO3 independent and resistant to mCPSF6-358, a truncated splicing factor that prevents HIV-1 nuclear import. Using a complementation assay based on 293T cells that express a TNPO3-targeted short hairpin RNA (shRNA), we showed that the Drosophila TNPO3 homologue can substitute for its human counterpart and that it mapped a key functional domain of TNPO3 to the carboxy-terminal cargo-binding domain. Within the cargo-binding domain, two hydrophobic motifs were required for TNPO3-dependent infection. The mutated TNPO3 proteins maintained their ability to localize to the nucleus, suggesting that their inability to restore lentivirus infection resulted from an inability to bind to a host or viral cargo protein.

Survival of verapamil-poisoned rats treated with triiodothyronine.

Life-threatening toxicity due to calcium channel blocker ingestion is commonly encountered by emergency medicine physicians and toxicologists. Despite a vast array of research on its treatment, results have proven inconsistent. The goal of this study is to evaluate potential vasopressor effects of triiodothyronine (T3) in rats poisoned with verapamil. Following anesthesia and intubation, ten Sprague-Dawley rats were given intravenous verapamil infusion of 10 mg/kg/h. This dose was titrated until a mean arterial pressure (MAP) of 50-55 mmHg was achieved and maintained for a period of at least 5 min. The verapamil infusion was then maintained at that rate. Five rats were randomized to receive a T3 bolus of 0.4 mcg/kg preceding an infusion of 1.5 mcg/kg/day which was doubled every 2 min until any of the following endpoints: systolic blood pressure of 100 mmHg, an elapsed time of 60 min, or death. The other five received an equal volume of normal saline solution. The primary outcome measure was survival with secondary outcomes of MAP and heart rate. The T3 group did have a slightly longer, yet not statistically significant, average time to cessation of electrical activity-30.0 +/- 14.4 min versus 23.8 +/- 9.5 min in the placebo group. Average MAP decreased nearly identically in the two groups. Heart rates were not reliable indicators of toxicity in this rat model as there was little decrease until immediately prior to death in most animals. Despite significant variability in toxicity among individual animals, no statistically significant difference in survival time, heart rate, or MAP was found between groups treated with T3 and those receiving saline.

Ethyl pyruvate enhances intra-resuscitation hemodynamics in prolonged ventricular fibrillation arrest.

AIMS: As the duration of untreated cardiac arrest increases, the effectiveness of standard therapies declines, and may be more harmful than helpful. We investigated the hemodynamic, metabolic and anti-inflammatory effects of Ringer's ethyl pyruvate solution (REPS) versus Ringer's solution (RS) in the acute model of prolonged porcine arrest. METHODS: Seventeen mixed-breed swine were induced into ventricular fibrillation (VF) and left untreated for 8min. CPR was begun using a mechanical chest compression device at a rate of 100 per minute. At the onset of CPR, animals were randomly assigned to treatment with either 25mL/kg of RS or 25mL/kg of REPS containing 40mg/kg of ethyl pyruvate, infused over 5min in blinded fashion. CPR continued with administration of a drug cocktail at 2min and the first rescue shock was delivered at minute 13 of VF. Animals having ROSC were supported with standardized care for 2h. RESULTS: Both groups had 100% ROSC and 100% 2-h survival. The REPS group exhibited higher median CPP (27.3mmHg) than the control group (16.5mmHg) by 3min of CPR, which continued throughout the duration of CPR (p=0.02). The median time to hypotension following ROSC was 9.64min in the REPS group and 7.25min in controls (p=0.04) and there was a non-significant trend of decreased use of vasopressors for the duration of resuscitation. There was no difference in systemic or cerebral metabolism between groups. There were non-significant trends of decreased IL-6, increased Il-10 and decreased mesenteric bacterial colony growth in those treated with REPS when compared to RS. CONCLUSIONS: The administration of REPS with CPR significantly improved intra- and post-resuscitation hemodynamics in this swine model of prolonged cardiac arrest, but did not definitely change the metabolic or inflammatory profile during the acute resuscitation period.

Lipid emulsion combined with epinephrine and vasopressin does not improve survival in a swine model of bupivacaine-induced cardiac arrest.

BACKGROUND: This study sought to evaluate the efficacy of lipid emulsion in reversing bupivacaine-induced cardiovascular collapse when added to a resuscitation protocol that included the use of epinephrine and vasopressin. METHODS: After induction of general anesthesia and instrumentation, 19 mixed-breed domestic swine had cardiovascular collapse induced by an intravenous bolus of 10 mg/kg bupivacaine. After 5 min of resuscitation including chest compressions, epinephrine (100 microg/kg) and vasopressin (1.5 U/kg), animals were randomized to receive either a bolus of 20% lipid emulsion (4 ml/kg) followed by a continuous infusion (0.5 ml x kg(-1) x min(-1)) or an equal volume of saline. Investigators were blinded to the treatment assignment. The primary endpoint was return of spontaneous circulation (mean arterial pressure of at least 60 mmHg for at least 1 min). RESULTS: Treatment groups were similar with respect to baseline measurements of weight, sex, and hemodynamic and metabolic variables. The rates of return of spontaneous circulation were similar between groups: (3 of 10) in the lipid group and 4 of 9 in the saline group (P = 0.65). Total serum bupivacaine concentrations were higher in the lipid group at the 10-min timepoint (mean +/- SEM: 23.13 +/- 5.37 ng/ml vs. 15.33 +/- 4.04 ng/ml, P = 0.004). More norepinephrine was required in the lipid group compared to the saline group to maintain a mean arterial pressure above 60 mmHg during the 60-min survival period (mean +/- SEM: 738.6 +/- 94.4 vs.. 487.3 +/- 171.0 microg). CONCLUSIONS: In this swine model, lipid emulsion did not improve rates of return of spontaneous circulation after bupivacaine-induced cardiovascular collapse.

Association of intramyocardial high energy phosphate concentrations with quantitative measures of the ventricular fibrillation electrocardiogram waveform.

BACKGROUND: Quantitative measures of the ventricular fibrillation (VF) electrocardiogram (ECG) have been correlated with the success of rescue shocks, making them ideal measures for guiding resuscitative interventions. Correlation of intramyocardial energy stores with the change in quantitative VF ECG measures would provide mechanistic insight into their utility. We sought to investigate the relationship between intramyocardial energy stores and four quantitative ECG measures. METHODS: Eighteen mixed-breed, domestic swine were sedated, anaesthetized and paralyzed. Swine were block randomized into three groups receiving 5, 10, or 15 min of untreated VF. Thoracotomy was performed and the heart was delivered. VF was induced by a 100 mA transthoracic shock while ECG was recorded. Biopsies of myocardial tissue were taken from the left and right ventricles after the prescribed duration of VF. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) concentrations in the tissue samples were measured. ECG data immediately prior to each biopsy were analyzed by each of four quantitative ECG methods: Scaling Exponent (ScE), Median Slope (MS), Amplitude Spectrum Area (AMSA), and logarithm of the Absolute Correlation (LAC). ATP and ADP concentrations of VF duration groups were compared. ATP and ADP concentrations were regressed against each quantitative ECG measure. RESULTS: ATP concentrations differed between VF duration groups, but ADP concentrations differed only between 5 and 10 min groups. A significant association existed between ATP and three quantitative measures--ScE, MS, and AMSA--but no significant relationship was found for ADP. CONCLUSION: Intramyocardial ATP levels correlate with quantitative measures of the ECG during ventricular fibrillation.

Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase.

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses.

Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation.

BACKGROUND: Hypothermia has been shown to improve survival and neurological outcomes for ventricular fibrillation (VF) cardiac arrest. The electrophysiological mechanisms of hypothermia are not well-understood, nor are the effects of beginning cooling during the resuscitation. METHODS AND RESULTS: We hypothesized that inducing hypothermia prior to the onset of VF would slow the deleterious changes seen in the ECG during VF and that inducing hypothermia at the start of resuscitation would increase the rates of ROSC and short-term survival in a porcine model of prolonged VF. We randomly assigned 42 domestic swine (27.2+/-2.3 kg) to either pretreatment with hypothermia before induction of VF (PRE), normothermic resuscitation (NORM) or intra-resuscitation hypothermia (IRH). During anesthesia, animals were instrumented via femoral cutdown. Lead II ECG was recorded continuously. PRE animals were cooled before the induction of VF, with a rapid infusion of 4 degrees normal saline (30mL/kg). VF was induced electrically, left untreated for 8min, then mechanical CPR began. During CPR the NORM animals got 30mL/kg body-temperature saline and the IRH animals got 30mL/kg 4 degrees saline. In all groups first rescue shocks were delivered after 13min of VF. We calculated the VF scaling exponent (ScE) for the entire 8min period (compared using GEE). ROSC and survival were compared with Fisher's exact test. Mean temperature in degrees C at the onset of VF was PRE=34.7 degrees (+/-0.8), NORM=37.8 (+/-0.9), and IRH=37.9 (+/-0.9). The ScE values over time were significantly lower after 8min in the PRE group (p=0.02). ROSC: PRE=10/14 (71%), NORM=6/14 (43%) and IRH=12/14 (86%); p for IRH vs. NORM=0.02. Survival: PRE=9/14 (64%), NORM=5/14 (36%), IRH 8/14 (57%). CONCLUSION: Hypothermia slowed the decay of the ECG waveform during prolonged VF. IRH improved ROSC but not short-term survival compared to NORM. It is possible to rapidly induce mild hypothermia during CPR using an IV infusion of ice-cold saline.

Brain-derived neurotrophic factor does not improve recovery after cardiac arrest in rats.

Increased brain-derived neurotrophic factor (BDNF) levels and extracellular-signal regulated kinase (ERK) signaling are associated with reduced brain injury after cerebral ischemia. In particular, mild hypothermia after cardiac arrest increases BDNF and ERK signaling. This study tested whether intracerebroventricular infusions (0.025 microg/h x 3 days) of BDNF also improved recovery of rats resuscitated from cardiac arrest and maintained at 37 degrees C. BDNF infusions initiated at the time of cardiac arrest did not alter survival, neurological recovery, or histological injury. Separate experiments confirmed that BDNF infusions increased tissue levels of BDNF. However, these infusions did not increase ERK activation in hippocampus. These data suggest that increased BDNF levels are not sufficient to explain the beneficial effects of mild hypothermia after cardiac arrest, and that exogenous BDNF administration does not increase extracellular ERK signaling.

Hypothermia after cardiac arrest does not alter serum inflammatory markers.

OBJECTIVE: Hypothermia improves survival and neurologic recovery after cardiac arrest. Cardiac arrest also triggers release of cytokines and inflammatory molecules, and it is unknown whether therapeutic hypothermia alters this inflammatory response. This study tested whether therapeutic hypothermia altered levels of inflammatory markers in serum. DESIGN: Prospective, randomized study. SETTING: University research laboratory. SUBJECTS: Adult, male, Sprague-Dawley rats. INTERVENTIONS: Halothane-anesthetized rats were subjected to 8 mins of asphyxial cardiac arrest and resuscitation. Rat temperature was controlled at 37 degrees C throughout the experiment (normothermia) or reduced to 33 degrees C between 1 and 24 hrs after cardiac arrest (hypothermia). Serum cytokines were measured at baseline, 0.5, 1, 3, 6, 12, and 24 hrs after resuscitation using multiplex analyzer or enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Hypothermic rats showed improved neurologic recovery at 12 and 24 hrs. Serum levels of tumor necrosis factor-alpha; macrophage inflammatory protein-1alpha; growth-related oncogene/keratinocyte chemokine; interleukin-2, -9, and -10; monocyte chemotactic protein-1; leptin; and intracellular adhesion molecule-1 increased over time, and the levels of interleukin-18 declined over time. No temporal trends in other molecules were detected. Levels of these molecules did not differ between temperature groups during the hypothermia phase (1-24 hrs). CONCLUSIONS: These data suggest that altering the inflammatory response after cardiac arrest is not necessary for the beneficial effects of hypothermia. These data do not support a specific role of circulating cytokines in the neurologic injury after cardiac arrest.

Increasing CPR duration prior to first defibrillation does not improve return of spontaneous circulation or survival in a swine model of prolonged ventricular fibrillation.

INTRODUCTION: The optimum duration of cardiopulmonary resuscitation (CPR) prior to first rescue shock is unknown. Clinical trials have used 90 and 180 s. Neither of these durations may be optimal. We sought to determine the optimum duration of CPR prior to first defibrillation attempt and whether this varied depending on the duration of ventricular fibrillation (VF). In this porcine model of basic life support, our outcomes were rates of return of spontaneous circulation (ROSC), survival, and coronary perfusion pressure (CPP). METHODS: We anesthetized and instrumented 45 swine and then induced VF. After 5 or 8 min of untreated VF, we randomized the swine to mechanical CPR for 90, 180, or 300 s. A single rescue shock (150 J biphasic) was then administered. If this shock failed, 2 min of mechanical CPR were completed prior to the next rescue shock. CPP was calculated for each 30s epoch. ROSC was defined as a blood pressure >80 mmHg sustained for 60s. Survival was defined as sustained ROSC for 20 min. Data were analyzed with descriptive statistics, Fisher's exact test, and ANOVA. RESULTS: In the 5 min VF cohort, the rate of ROSC did not differ between the three groups (90 s: 25%; 180 s: 38%; 300 s: 38%, p>.05). Survival rates did not differ (90 s: 25%; 180 s: 25%; 300 s: 25%, p>0.05). In the 8 min VF cohort, no animals experienced ROSC or survival. CPP were calculated by 30s epoch and did not differ between the three groups (p>0.05). CPPs decline after 180 s of CPR. CONCLUSIONS: ROSC and survival were equivalent regardless of VF duration and CPR duration. When CPR begins late, CPPs are low, stressing the importance of early CPR. We do not recommend 300 s of CPR unless a defibrillator is unavailable.

The effect of adenosine A1 receptor antagonism on return of spontaneous circulation and short-term survival in prolonged ventricular fibrillation.

BACKGROUND: Endogenous adenosine (ADO) is cardioprotective during ischemia and its myocardial concentration increases during untreated ventricular fibrillation (VF). We have previously shown that ADO A1 receptor (ADOA1R) antagonism hastens the time-dependent decay in VF waveform morphology during the circulatory phase of cardiac arrest. OBJECTIVE: To determine the effect of ADOA1R antagonism on ROSC and short-term survival in prolonged VF. METHODS: Thirty-six swine were assigned by block randomization to one of three groups: a group that received only vehicle (CONTROL), an ADOA1R antagonist pretreatment group (PRE), and a group that was given ADOA1R antagonist during resuscitation (DURING). The animals were instrumented under anesthesia, and ADOA1R antagonist or vehicle, per group assignment, was infused 5 minutes prior to VF induction. At minute 8 of untreated VF, chest compression with ventilation was initiated and a standard drug cocktail, with ADOA1R antagonist or vehicle, was given. The first rescue shock (150 J biphasic) was delivered after 11 minutes of VF. Proportions with 95% confidence intervals (CIs) were calculated for the two outcome measures. RESULTS: The baseline characteristics and chemistry values for the three groups were mathematically the same. The DURING group had a greater proportion of female animals (seven of 12) in comparison with the CONTROL group (two of 12) (p=0.03). ADOA1R antagonism hastened the decay of VF as previously demonstrated, but the rate of ROSC was the same for all groups: CONTROL=seven of 12, PRE=six of 12, and DURING=seven of 12. There were also no differences in short-term survival: CONTROL=four of 12, PRE=five of 12, and DURING=seven of 12. CONCLUSIONS: In this study, ADOA1R antagonism had no effect on outcome whether given before induction of VF or upon resuscitation after 8 minutes of untreated VF. The role of endogenous ADO in prolonged VF remains unclear.