Peritoneal sepsis caused by Escherichia coli triggers brainstem inflammation and alters the function of sympatho-respiratory control circuits.

BACKGROUND: Sepsis has a high mortality rate due to multiple organ failure. However, the influence of peripheral inflammation on brainstem autonomic and respiratory circuits in sepsis is poorly understood. Our working hypothesis is that peripheral inflammation affects central autonomic circuits and consequently contributes to multiorgan failure in sepsis. METHODS: In an Escherichia coli (E. coli)-fibrin clot model of peritonitis, we first recorded ventilatory patterns using plethysmography before and 24 h after fibrin clot implantation. To assess whether peritonitis was associated with brainstem neuro-inflammation, we measured cytokine and chemokine levels in Luminex assays. To determine the effect of E. coli peritonitis on brainstem function, we assessed sympatho-respiratory nerve activities at baseline and during brief (20 s) hypoxemic ischemia challenges using in situ-perfused brainstem preparations (PBPs) from sham or infected rats. PBPs lack peripheral organs and blood, but generate vascular tone and in vivo rhythmic activities in thoracic sympathetic (tSNA), phrenic and vagal nerves. RESULTS: Respiratory frequency was greater (p < 0.001) at 24 h post-infection with E. coli than in the sham control. However, breath-by-breath variability and total protein in the BALF did not differ. IL-1ß (p < 0.05), IL-6 (p < 0.05) and IL-17 (p < 0.04) concentrations were greater in the brainstem of infected rats. In the PBP, integrated tSNA (p < 0.05) and perfusion pressure were greater (p < 0.001), indicating a neural-mediated pathophysiological high sympathetic drive. Moreover, respiratory frequency was greater (p < 0.001) in PBPs from infected rats than from sham rats. Normalized phase durations of inspiration and expiration were greater (p < 0.009, p < 0.015, respectively), but the post-inspiratory phase (p < 0.007) and the breath-by-breath variability (p < 0.001) were less compared to sham PBPs. Hypoxemic ischemia triggered a biphasic response, respiratory augmentation followed by depression. PBPs from infected rats had weaker respiratory augmentation (p < 0.001) and depression (p < 0.001) than PBPs from sham rats. In contrast, tSNA in E. coli-treated PBPs was enhanced throughout the entire response to hypoxemic ischemia (p < 0.01), consistent with sympathetic hyperactivity. CONCLUSION: We show that peripheral sepsis caused brainstem inflammation and impaired sympatho-respiratory motor control in a single day after infection. We conclude that central sympathetic hyperactivity may impact vital organ systems in sepsis.

Brainstem inflammation modulates the ventilatory pattern and its variability after acute lung injury in rodents.

KEY POINTS: Compared with sham rats, rats a week after acute lung injury (ALI) express more pro-inflammatory cytokines in their brainstem respiratory control nuclei, exhibit a higher respiratory frequency (fR) and breathe with a more predictable pattern. These characteristics of the respiratory pattern persist in in situ preparations even after minimizing pulmonary and chemo-afferent inputs. Interleukin (IL)-1ß microinjected in the nucleus tractus solitarii increases fR and the predictability of the ventilatory pattern similar to rats with ALI. Intracerebroventricular infusion of indomethacin, an anti-inflammatory drug, mitigates the effect of ALI on fR and ventilatory pattern variability. We conclude that changes in the ventilatory pattern after ALI result not only from sensory input due to pulmonary damage and dysfunction but also from neuro-inflammation. ABSTRACT: Acute lung injury (ALI) increases respiratory rate (fR) and ventilatory pattern variability (VPV), but also evokes peripheral and central inflammation. We hypothesized that central inflammation has a role in determining the ventilatory pattern after ALI. In rat pups, we intratracheally injected either bleomycin to induce ALI or saline as a sham control. One week later, we recorded the ventilatory pattern of the rat pups using flow-through plethysmography, then formed in situ preparations from these pups and recorded their 'fictive' patterns from respiratory motor nerves. Compared with the ventilatory pattern of the sham rat pups, injured rat pups had increased fR and predictability. Surprisingly, the fictive patterns of the in situ preparations from ALI pups retained these characteristics despite removing their lungs to eliminate pulmonary sensory inputs and perfusing them with hyperoxic artificial cerebral spinal fluid to minimize peripheral chemoreceptor input. Histological processing revealed increased immunoreactivity of the pro-inflammatory cytokine Interleukin-1ß (IL-1ß) in the nucleus tractus solitarii (nTS) from ALI but not sham rats. In subsequent experiments, we microinjected IL-1ß in the nTS bilaterally in anaesthetized naïve adult rats, which increased fR and predictability of ventilatory pattern variability (VPV) after 2 h. Finally, we infused indomethacin intracerebroventricularly during the week of survival after ALI. This did not affect sham rats, but mitigated changes in fR and VPV in ALI rats. We conclude that neuro-inflammation has an essential role in determining the ventilatory pattern of ALI rats.

Peripheral-to-central immune communication at the area postrema glial-barrier following bleomycin-induced sterile lung injury in adult rats.

The pathways for peripheral-to-central immune communication (P â†’ C I-comm) following sterile lung injury (SLI) are unknown. SLI evokes systemic and central inflammation, which alters central respiratory control and viscerosensory transmission in the nucleus tractus solitarii (nTS). These functional changes coincide with increased interleukin-1 beta (IL-1ß) in the area postrema, a sensory circumventricular organ that connects P â†’ C I-comm to brainstem circuits that control homeostasis. We hypothesize that IL-1ß and its downstream transcriptional target, cyclooxygenase-2 (COX-2), mediate P â†’ C I-comm in the nTS. In a rodent model of SLI induced by intratracheal bleomycin (Bleo), the sigh frequency and duration of post-sigh apnea increased in Bleo- compared to saline- treated rats one week after injury. This SLI-dependent change in respiratory control occurred concurrently with augmented IL-1ß and COX-2 immunoreactivity (IR) in the funiculus separans (FS), a barrier between the AP and the brainstem. At this barrier, increases in IL-1ß and COX-2 IR were confined to processes that stained for glial fibrillary acidic protein (GFAP) and that projected basolaterally to the nTS. Further, FS radial-glia did not express TNF-α or IL-6 following SLI. To test our hypothesis, we blocked central COX-1/2 activity by intracerebroventricular (ICV) infusion of Indomethacin (Ind). Continuous ICV Ind treatment prevented Bleo-dependent increases in GFAP + and IL-1ß + IR, and restored characteristics of sighs that reset the rhythm. These data indicate that changes in sighs following SLI depend partially on activation of a central COX-dependent P â†’ C I-comm via radial-glia of the FS.

INSMA: An integrated system for multimodal data acquisition and analysis in the intensive care unit.

Modern intensive care units (ICU) are equipped with a variety of different medical devices to monitor the physiological status of patients. These devices can generate large amounts of multimodal data daily that include physiological waveform signals (arterial blood pressure, electrocardiogram, respiration), patient alarm messages, numeric vitals data, etc. In order to provide opportunities for increasingly improved patient care, it is necessary to develop an effective data acquisition and analysis system that can assist clinicians and provide decision support at the patient bedside. Previous research has discussed various data collection methods, but a comprehensive solution for bedside data acquisition to analysis has not been achieved. In this paper, we proposed a multimodal data acquisition and analysis system called INSMA, with the ability to acquire, store, process, and visualize multiple types of data from the Philips IntelliVue patient monitor. We also discuss how the acquired data can be used for patient state tracking. INSMA is being tested in the ICU at University Hospitals Cleveland Medical Center.

Lung-injury depresses glutamatergic synaptic transmission in the nucleus tractus solitarii via discrete age-dependent mechanisms in neonatal rats.

Transition periods (TPs) are brief stages in CNS development where neural circuits can exhibit heightened vulnerability to pathologic conditions such as injury or infection. This susceptibility is due in part to specialized mechanisms of synaptic plasticity, which may become activated by inflammatory mediators released under pathologic conditions. Thus, we hypothesized that the immune response to lung injury (LI) mediated synaptic changes through plasticity-like mechanisms that depended on whether LI occurred just before or after a TP. We studied the impact of LI on brainstem 2nd-order viscerosensory neurons located in the nucleus tractus solitarii (nTS) during a TP for respiratory control spanning (postnatal day (P) 11-15). We injured the lungs of Sprague-Dawley rats by intratracheal instillation of Bleomycin (or saline) just before (P9-11) or after (P17-19) the TP. A week later, we prepared horizontal slices of the medulla and recorded spontaneous and evoked excitatory postsynaptic currents (sEPSCs/eEPSCs) in vitro from neurons in the nTS that received monosynaptic glutamatergic input from the tractus solitarii (TS). In rats injured before the TP (pre-TP), neurons exhibited blunted sEPSCs and TS-eEPSCs compared to controls. The decreased TS-eEPSCs were mediated by differences in postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid receptors (AMPAR). Specifically, compared to controls, LI rats had more Ca2+-impermeable AMPARs (CI-AMPARs) as indicated by: 1) the absence of current-rectification, 2) decreased sensitivity to polyamine, 1-Naphthyl-acetyl-spermine-trihydrochloride (NASPM) and 3) augmented immunoreactive staining for the CI-AMPAR GluA2. Thus, pre-TP-LI acts postsynaptically to blunt glutamatergic transmission. The neuroimmune response to pre-TP-LI included microglia hyper-ramification throughout the nTS. Daily intraperitoneal administration of minocycline, an inhibitor of microglial/macrophage function prevented hyper-ramification and abolished the pre-TP-LI evoked synaptic changes. In contrast, rat-pups injured after the TP (post-TP) exhibited microglia hypo-ramification in the nTS and had increased sEPSC amplitudes/frequencies, and decreased TS-eEPSC amplitudes compared to controls. These synaptic changes were not associated with changes in CI-AMPARs, and instead involved greater TS-evoked use-dependent depression (reduced paired pulse ratio), which is a hallmark of presynaptic plasticity. Thus we conclude that LI regulates the efficacy of TS → nTS synapses through discrete plasticity-like mechanisms that are immune-mediated and depend on whether the injury occurs before or after the TP for respiratory control.

Co-registration of pre-operative CT with ex vivo surgically excised ground glass nodules to define spatial extent of invasive adenocarcinoma on in vivo imaging: a proof-of-concept study.

OBJECTIVE: To develop an approach for radiology-pathology fusion of ex vivo histology of surgically excised pulmonary nodules with pre-operative CT, to radiologically map spatial extent of the invasive adenocarcinomatous component of the nodule. METHODS: Six subjects (age: 75 ± 11 years) with pre-operative CT and surgically excised ground-glass nodules (size: 22.5 ± 5.1 mm) with a significant invasive adenocarcinomatous component (>5 mm) were included. The pathologist outlined disease extent on digitized histology specimens; two radiologists and a pulmonary critical care physician delineated the entire nodule on CT (in-plane resolution: <0.8 mm, inter-slice distance: 1-5 mm). We introduced a novel reconstruction approach to localize histology slices in 3D relative to each other while using CT scan as spatial constraint. This enabled the spatial mapping of the extent of tumour invasion from histology onto CT. RESULTS: Good overlap of the 3D reconstructed histology and the nodule outlined on CT was observed (65.9 ± 5.2%). Reduction in 3D misalignment of corresponding anatomical landmarks on histology and CT was observed (1.97 ± 0.42 mm). Moreover, the CT attenuation (HU) distributions were different when comparing invasive and in situ regions. CONCLUSION: This proof-of-concept study suggests that our fusion method can enable the spatial mapping of the invasive adenocarcinomatous component from 2D histology slices onto in vivo CT. KEY POINTS: • 3D reconstructions are generated from 2D histology specimens of ground glass nodules. • The reconstruction methodology used pre-operative in vivo CT as 3D spatial constraint. • The methodology maps adenocarcinoma extent from digitized histology onto in vivo CT. • The methodology potentially facilitates the discovery of CT signature of invasive adenocarcinoma.

Information technology in critical care: review of monitoring and data acquisition systems for patient care and research.

There is a broad consensus that 21st century health care will require intensive use of information technology to acquire and analyze data and then manage and disseminate information extracted from the data. No area is more data intensive than the intensive care unit. While there have been major improvements in intensive care monitoring, the medical industry, for the most part, has not incorporated many of the advances in computer science, biomedical engineering, signal processing, and mathematics that many other industries have embraced. Acquiring, synchronizing, integrating, and analyzing patient data remain frustratingly difficult because of incompatibilities among monitoring equipment, proprietary limitations from industry, and the absence of standard data formatting. In this paper, we will review the history of computers in the intensive care unit along with commonly used monitoring and data acquisition systems, both those commercially available and those being developed for research purposes.

Do heart and respiratory rate variability improve prediction of extubation outcomes in critically ill patients?

INTRODUCTION: Prolonged ventilation and failed extubation are associated with increased harm and cost. The added value of heart and respiratory rate variability (HRV and RRV) during spontaneous breathing trials (SBTs) to predict extubation failure remains unknown. METHODS: We enrolled 721 patients in a multicenter (12 sites), prospective, observational study, evaluating clinical estimates of risk of extubation failure, physiologic measures recorded during SBTs, HRV and RRV recorded before and during the last SBT prior to extubation, and extubation outcomes. We excluded 287 patients because of protocol or technical violations, or poor data quality. Measures of variability (97 HRV, 82 RRV) were calculated from electrocardiogram and capnography waveforms followed by automated cleaning and variability analysis using Continuous Individualized Multiorgan Variability Analysis (CIMVA™) software. Repeated randomized subsampling with training, validation, and testing were used to derive and compare predictive models. RESULTS: Of 434 patients with high-quality data, 51 (12%) failed extubation. Two HRV and eight RRV measures showed statistically significant association with extubation failure (P <0.0041, 5% false discovery rate). An ensemble average of five univariate logistic regression models using RRV during SBT, yielding a probability of extubation failure (called WAVE score), demonstrated optimal predictive capacity. With repeated random subsampling and testing, the model showed mean receiver operating characteristic area under the curve (ROC AUC) of 0.69, higher than heart rate (0.51), rapid shallow breathing index (RBSI; 0.61) and respiratory rate (0.63). After deriving a WAVE model based on all data, training-set performance demonstrated that the model increased its predictive power when applied to patients conventionally considered high risk: a WAVE score >0.5 in patients with RSBI >105 and perceived high risk of failure yielded a fold increase in risk of extubation failure of 3.0 (95% confidence interval (CI) 1.2 to 5.2) and 3.5 (95% CI 1.9 to 5.4), respectively. CONCLUSIONS: Altered HRV and RRV (during the SBT prior to extubation) are significantly associated with extubation failure. A predictive model using RRV during the last SBT provided optimal accuracy of prediction in all patients, with improved accuracy when combined with clinical impression or RSBI. This model requires a validation cohort to evaluate accuracy and generalizability. TRIAL REGISTRATION: ClinicalTrials.gov NCT01237886. Registered 13 October 2010.

Effects of environment light during sleep on autonomic functions of heart rate and breathing.

PURPOSE: Poor sleep hygiene including sleeping in the daytime or with the lights on at night is discovered during the assessment of many sleep disorders including sleep apnea. The aim of this study was to investigate whether environmental light affected autonomic control of heart rate, sleep-disordered breathing (SDB), and/or breathing patterning. METHODS: Seventeen non-obese healthy volunteers without witnessed snoring and apneas were recruited. Studies were performed at home using a type 3 portable monitor combined with actigraphy for sleep-wake timing, using a randomly assigned, crossover between dark, or 1,000 lx of fluorescent lighting environment. The outcomes were low-frequency power divided by high-frequency power (LF/HF ratio) in the analysis of heart rate variability, the apnea-hypopnea index (AHI), and ventilatory pattern variability before and after sleep onset between environments. RESULTS: The LF/HF ratio and AHI were both significantly higher in light as compared to dark. Before sleep onset, the coefficient of variation (CV) for breath-to-breath tidal volume representing breathing irregularity tended to be higher in light than in dark environment. The CV values for tidal volume after sleep onset were significantly decreased compared with before sleep onset in both sleep environments. Mutual information of the ventilatory pattern was significantly lower before sleep onset than after sleep onset, only in the light environment. CONCLUSIONS: Sleeping in the light has effects like that of a stressor as it is associated with neuroexcitation, SDB, and resting breathing irregularity in healthy volunteers. These findings may be relevant to many sleep disorders associated with poor sleep hygiene.

Persistent glossopharyngeal nerve respiratory discharge patterns after ponto-medullary transection.

Shape and size of the nasopharyngeal airway is controlled by muscles innervated facial, glossopharyngeal, vagal, and hypoglossal cranial nerves. Contrary to brainstem networks that drive facial, vagal and hypoglossal nerve activities (FNA, VNA, HNA) the discharge patterns and origins of glossopharyngeal nerve activity (GPNA) remain poorly investigated. Here, an in situ perfused brainstem preparation (n=19) was used for recordings of GPNA in relation to phrenic (PNA), FNA, VNA and HNA. Brainstem transections were performed (n=10/19) to explore the role of pontomedullary synaptic interactions in generating GPNA. GPNA generally mirrors FNA and HNA discharge patterns and displays pre-inspiratory activity relative to the PNA, followed by robust inspiratory discharge in coincidence with PNA. Postinspiratory (early expiratory) discharge was, contrary to VNA, generally absent in FNA, GPNA or HNA. As described previously FNA and HNA discharge was virtually eliminated after pontomedullary transection while an apneustic inspiratory motor discharge was maintained in PNA, VNA and GPNA. After brainstem transection GPNA displayed an increased tonic activity starting during mid-expiration and thus developed prolonged pre-inspiratory activity compared to control. In conclusion respiratory GPNA reflects FNA and HNA which implies similar function in controlling upper airway patency during breathing. That GPNA preserved its pre-inspiratory/inspiratory discharge pattern in relation PNA after pontomedullary transection suggest that GPNA premotor circuits may have a different anatomical distribution compared HNA and FNA and thus may therefore hold a unique role in preserving airway patency.

Breathing irregularity during wakefulness associates with CPAP acceptance in sleep apnea.

PURPOSE: Individuals have different breathing patterns at rest, during wakefulness, and during sleep, and patients with sleep apnea are no different. The hypothesis for this study was that breathing irregularity during wakefulness associates with CPAP acceptance in obstructive sleep apnea (OSA). METHODS: From a 2007-2010-database of patients with a diagnostic polysomnography (PSG) and prescribed CPAP (n = 380), retrospectively, 66 patients who quit CPAP treatment at 6 months were identified. Among them, 27 OSA patients quit despite having no side effects for discontinuing CPAP (Group A) and were compared to a matched group (age, body mass index, and apnea-hypopnea index) with good 6-month CPAP adherence (Group B; n = 21). Five minutes of respiratory signal during wakefulness at the initial PSG were extracted from respiratory inductance plethysmography recordings, and measured in a blinded fashion. The coefficients of variation (CV) for the breath-to-breath inspiration time (T i), expiration time (T e), T i + T e (T tot), and relative tidal volume, as well as an independent information theory-based metric of signal pattern variability (mutual information) were compared between groups. RESULTS: The CV for tidal volume was significantly greater (p = 0.001), and mutual information was significantly lower (p = 0.041) in Group A as compared to Group B. CONCLUSIONS: Differences in two independent measures of breathing irregularity correlated with CPAP rejection in OSA patients without nasal symptoms or comorbidity. Prospective studies of adherence should examine traits of breathing stability.

The use of heart rate variability for the early detection of treatable complications after aneurysmal subarachnoid hemorrhage.

High-grade aneurysmal subarachnoid hemorrhage patients are monitored in the ICU for up to 21 days, as they are at risk for complications such as vasospasm of cerebral arteries, cardiac arrhythmias and neurogenic stress cardiomyopathy. The diagnosis of these treatable complications is often delayed by the limitations of monitoring capabilities. We applied computational analysis to a cohort of 24 aneurysmal subarachnoid hemorrhage patients, to identify heart rate variability and ECG frequency profiles that may be potential biomarkers of severe vasospasm, reversible cardiomyopathy and death.

Cannabinoid Receptor mRNA Expression in Central and Peripheral Tissues in a Rodent Model of Peritonitis.

Introduction: Our laboratory investigates changes in the respiratory pattern during systemic inflammation in various rodent models. The endogenous cannabinoid system (ECS) regulates cytokine production and mitigates inflammation. Inflammation not only affects cannabinoid (CB) 1 and CB2 receptor gene expression (Cnr1 and Cnr2), but also increases the predictability of the ventilatory pattern. Objectives: Our primary objective was to track ventilatory pattern variability and transcription of Cnr1 and Cnr2 mRNA, and of Il1b, Il6, and tumor necrosis factor-alpha (Tnfa) mRNAs at multiple time points in central and peripheral tissues during systemic inflammation induced by peritonitis. Methods: In male Sprague Dawley rats (n=24), we caused peritonitis by implanting a fibrin clot containing either 0 or 25×106 Escherichia coli intraperitoneally. We recorded breathing with whole-animal plethysmography at baseline and 1 h before euthanasia. We euthanized the rats at 3, 6, or 12 h after inoculation and harvested the pons, medulla, lung, and heart for gene expression analysis. Results: With peritonitis, Cnr1 mRNA more than Cnr2 mRNA was correlated to Il1b, Il6, and Tnfa mRNAs in medulla, pons, and lung and changed oppositely in the pons, medulla, and lung. These changes were associated with increased predictability of ventilatory pattern. Specifically, nonlinear complexity index correlated with increased Cnr1 mRNA in the pons and medulla, and coefficient of variation for cycle duration correlated with Cnr1 and Cnr2 mRNAs in the lung. Conclusion: The mRNAs for ECS receptors varied with time during the central and peripheral inflammatory response to peritonitis. These changes occurred in the brainstem, which contains the network that generates breathing pattern and thus, may participate in ventilatory pattern changes during systemic inflammation.

Dynamics of ventilatory pattern variability and Cardioventilatory Coupling during systemic inflammation in rats.

Introduction: Biometrics of common physiologic signals can reflect health status. We have developed analytics to measure the predictability of ventilatory pattern variability (VPV, Nonlinear Complexity Index (NLCI) that quantifies the predictability of a continuous waveform associated with inhalation and exhalation) and the cardioventilatory coupling (CVC, the tendency of the last heartbeat in expiration to occur at preferred latency before the next inspiration). We hypothesized that measures of VPV and CVC are sensitive to the development of endotoxemia, which evoke neuroinflammation. Methods: We implanted Sprague Dawley male rats with BP transducers to monitor arterial blood pressure (BP) and recorded ventilatory waveforms and BP simultaneously using whole-body plethysmography in conjunction with BP transducer receivers. After baseline (BSLN) recordings, we injected lipopolysaccharide (LPS, n = 8) or phosphate buffered saline (PBS, n =3) intraperitoneally on 3 consecutive days. We recorded for 4-6 h after the injection, chose 3 epochs from each hour and analyzed VPV and CVC as well as heart rate variability (HRV). Results: First, the responses to sepsis varied across rats, but within rats the repeated measures of NLCI, CVC, as well as respiratory frequency (fR), HR, BP and HRV had a low coefficient of variation, (<0.2) at each time point. Second, HR, fR, and NLCI increased from BSLN on Days 1-3; whereas CVC decreased on Days 2 and 3. In contrast, changes in BP and the relative low-(LF) and high-frequency (HF) of HRV were not significant. The coefficient of variation decreased from BSLN to Day 3, except for CVC. Interestingly, NLCI increased before fR in LPS-treated rats. Finally, we histologically confirmed lung injury, systemic inflammation via ELISA and the presence of the proinflammatory cytokine, IL-1ß, with immunohistochemistry in the ponto-medullary respiratory nuclei. Discussion: Our findings support that NLCI reflects changes in the rat's health induced by systemic injection of LPS and reflected in increases in HR and fR. CVC decreased over the course to the experiment. We conclude that NLCI reflected the increase in predictability of the ventilatory waveform and (together with our previous work) may reflect action of inflammatory cytokines on the network generating respiration.

Inhibiting Mycobacterium abscessus Cell Wall Synthesis: Using a Novel Diazabicyclooctane ß-Lactamase Inhibitor To Augment ß-Lactam Action.

Mycobacterium abscessus (Mab) infections are a growing menace to the health of many patients, especially those suffering from structural lung disease and cystic fibrosis. With multidrug resistance a common feature and a growing understanding of peptidoglycan synthesis in Mab, it is advantageous to identify potent ß-lactam and ß-lactamase inhibitor combinations that can effectively disrupt cell wall synthesis. To improve existing therapeutic regimens to address serious Mab infections, we evaluated the ability of durlobactam (DUR), a novel diazobicyclooctane ß-lactamase inhibitor to restore in vitro susceptibilities in combination with ß-lactams and provide a biochemical rationale for the activity of this compound. In cell-based assays, susceptibility of Mab subsp. abscessus isolates to amoxicillin (AMOX), imipenem (IMI), and cefuroxime (CXM) was significantly enhanced with the addition of DUR. The triple drug combinations of CXM-DUR-AMOX and IMI-DUR-AMOX were most potent, with MIC ranges of ≤0.06 to 1 µg/mL and an MIC50/MIC90 of ≤0.06/0.25 µg/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the ß-lactamase BlaMab with a relative Michaelis constant (Ki app) of 4 × 10-3 ± 0.8 × 10-3 µM and acylation rate (k2/K) of 1 × 107 M-1 s-1. Timed mass spectrometry captured stable formation of carbamoyl-enzyme complexes between DUR and LdtMab2-4 and Mab d,d-carboxypeptidase, potentially contributing to the intrinsic activity of DUR. Molecular modeling showed unique and favorable interactions of DUR as a BlaMab inhibitor. Similarly, modeling showed how DUR might form stable Michaelis-Menten complexes with LdtMab2-4 and Mab d,d-carboxypeptidase. The ability of DUR combined with amoxicillin or cefuroxime and imipenem to inactivate multiple targets such as d,d-carboxypeptidase and LdtMab2,4 supports new therapeutic approaches using ß-lactams in eradicating Mab. IMPORTANCE Durlobactam (DUR) is a potent inhibitor of BlaMab and provides protection of amoxicillin and imipenem against hydrolysis. DUR has intrinsic activity and forms stable acyl-enzyme complexes with LdtMab2 and LdtMab4. The ability of DUR to protect amoxicillin and imipenem against BlaMab and its intrinsic activity along with the dual ß-lactam target redundancy can explain the rationale behind the potent activity of this combination.

Heritability of abnormalities in cardiopulmonary coupling in sleep apnea: use of an electrocardiogram-based technique.

RATIONALE: Studies of the genetics of obstructive sleep apnea may be facilitated by identifying intermediate traits with high heritability that quantify etiological pathways, such as those related to respiratory control. Electrocardiogram (ECG)-based sleep spectrograms, measuring the coupling between respiratory modulation of ECG QRS-wave amplitude and heart rate variability, may provide measures of sleep state and ventilatory dynamics during sleep. We evaluated the familial aggregation of distinctive spectrographic biomarkers of unstable sleep, related to elevated-low frequency cardiopulmonary coupling (e-LFC), to assess their utility in genetic studies. METHODS: 622 participants from 137 families from the Cleveland Family Study underwent standardized polysomnography (PSG). From the ECG signal on the PSG, the interbeat interval time series and the corresponding ECG-derived respiratory signal were extracted, and the low frequency (0.01-0.1 Hz) component of their coupling was computed using a fully automated method. Narrow sense heritability of e-LFC was calculated using variance component methods. RESULTS: A spectral marker of abnormal low frequency cardiopulmonary coupling (e-LFC) demonstrated moderate correlation with apnea hypopnea index (AHI; r = 0.35, P < 0.0001). The heritability estimate for e-LFC, after adjusting for age and sex was 0.32 (P < 10-5) and remained unchanged after additionally adjusting for body mass index or AHI. In biological relatives of those with sleep apnea, a related marker of e-LFC was more prevalent than in controls (P = 0.05). CONCLUSIONS: Approximately 30% of the variability of e-LFC, measured from a continuous ECG during sleep, is explained by familial factors other than BMI. ECG-based spectrographic measures of cardiopulmonary coupling may provide novel phenotypes for characterizing subgroups of individuals with different propensities and genetic etiologies for sleep apnea or for other conditions associated with sleep fragmentation.

Ventilatory pattern variability as a biometric for severity of acute lung injury in rats.

We hypothesize that ventilatory pattern variability (VPV) varies with the magnitude of acute lung injury (ALI). In adult male rats, we instilled a low- or high- dose of bleomycin or saline (PBS) intratracheally. While representative samples of pulmonary tissue indicated graded lung injury, coefficient of variation (CV) of TTOT did not differ among the 3 groups. Broncho-alveolar lavage fluid (BALF), respiratory rate (fR), mutual information were greater in ALI than sham rats; but did not differ between bleomycin doses. However, nonlinear complexity index (NLCI), which is the difference in sample entropy between original and surrogate data sets was greater for high- versus low- dose; but did not differ between low-dose and sham groups. Further, NLCI correlated to an injury index based on protein concentration of BALF and failure to gain weight. Finally, Receiver Operator Curves (ROCs) indicated that both mutual information and NLCI had greater sensitivity and specificity than fR and CVTTOT in identifying ALI. Thus, nonlinear analyses of VPV can distinguish ALI and out performs fR as a biometric.

Acute lung injury in neonatal rats causes postsynaptic depression in nucleus tractus solitarii second-order neurons.

Acute Lung Injury (ALI) alters pulmonary reflex responses, in part due to changes in modulation within the lung and airway neuronal control networks. We hypothesized that synaptic efficacy of nucleus tractus solitarii (nTS) neurons, receiving input from lung, airway, and other viscerosensory afferent fibers, would decrease following ALI. Sprague Dawley neonatal rats (postnatal days 9-11) were given intratracheal installations of saline or bleomycin (a well-characterized model that reproduces the pattern of ALI) and then, one week later, in vitro slices were prepared for whole-cell and perforated whole-cell patch-clamp experiments (postnatal days 16-21). In preparations from ALI rats, 2nd-order nTS neurons had significantly decreased amplitudes of both spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs), compared to saline controls. Rise and decay times of sEPSCs were slower in whole-cell recordings from ALI animals. Similarly, the amplitude of tractus solitarii evoked EPSCs (TS-eEPSCs) were significantly lower in 2nd-order nTS neurons from ALI rats. Overall these results suggest the presence of postsynaptic depression at TS-nTS synapses receiving lung, airway, and other viscerosensory afferent tractus solitarii input after bleomycin-induced ALI.

AGTR2 absence or antagonism prevents cystic fibrosis pulmonary manifestations.

BACKGROUND: Pulmonary disease remains the primary cause of morbidity and mortality for individuals with cystic fibrosis (CF). Variants at a locus on the X-chromosome containing the type 2 angiotensin II receptor gene (AGTR2) were identified by a large GWAS as significantly associating with lung function in CF patients. We hypothesized that manipulating the angiotensin-signaling pathway may yield clinical benefit in CF. METHODS: Genetic subset analysis was conducted on a local CF cohort to extend the GWAS findings. Next, we evaluated pulmonary function in CF mice with a deleted AGTR2 gene, and in those who were given subcutaneous injections of PD123,319, a selective AGTR2 antagonist for 12 weeks beginning at weaning. RESULTS: The genetic subset analysis replicated the initial GWAS identified association, and confirmed the association of this locus with additional lung function parameters. Studies in genetically modified mice established that absence of the AGTR2 gene normalized pulmonary function indices in two independent CF mouse models. Further, we determined that pharmacologic antagonism of AGTR2 improved overall pulmonary function in CF mice to near wild-type levels. CONCLUSIONS: These results identify that reduced AGTR2 signaling is beneficial to CF lung function, and suggest the potential of manipulating the angiotensin-signaling pathway for treatment and/or prevention of CF pulmonary disease. Importantly, the beneficial effects were not CF gene mutation dependent, and were able to be reproduced with pharmacologic antagonism. As there are clinically approved drugs available to target the renin-angiotensin signaling system, these findings may be quickly translated to human clinical trials.

Comparative analysis of neonatal and adult rat carotid body responses to chronic intermittent hypoxia.

Previous studies suggest that carotid body responses to long-term changes in environmental oxygen differ between neonates and adults. In the present study we tested the hypothesis that the effects of chronic intermittent hypoxia (CIH) on the carotid body differ between neonates and adult rats. Experiments were performed on neonatal (1-10 days) and adult (6-8 wk) males exposed either to CIH (9 episodes/h; 8 h/day) or to normoxia. Sensory activity was recorded from ex vivo carotid bodies. CIH augmented the hypoxic sensory response (HSR) in both groups. The magnitude of CIH-evoked hypoxic sensitization was significantly greater in neonates than in adults. Seventy-two episodes of CIH were sufficient to evoke hypoxic sensitization in neonates, whereas as many as 720 CIH episodes were required in adults, suggesting that neonatal carotid bodies are more sensitive to CIH than adult carotid bodies. CIH-induced hypoxic sensitization was reversed in adult rats after reexposure to 10 days of normoxia, whereas the effects of neonatal CIH persisted into adult life (2 mo). Acute intermittent hypoxia (IH) evoked sensory long-term facilitation of the carotid body activity (sensory LTF, i.e., increased baseline neural activity following acute IH) in CIH-exposed adults but not in neonates. The effects of CIH were associated with hyperplasia of glomus cells in neonatal but not in adult carotid bodies. These observations demonstrate that responses to CIH differ between neonates and adults with regard to the magnitude of sensitization of HSR, susceptibility to CIH, induction of sensory LTF, reversibility of the responses, and morphological remodeling of the chemoreceptor tissue.