Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called “organoids,” to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems.

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We compared crural diaphragm electrical activity (EAdi) with transdiaphragmatic pressure (Pdi) during varying levels of pressure support ventilation (PS) in 13 intubated patients. With changing PS, we found no evidence for changes in neuromechanical coupling of the diaphragm. From lowest to highest PS (2 cm H(2)O +/- 4 to 20 cm H(2)O +/- 7), tidal volume increased from 430 ml +/- 180 to 527 ml +/- 180 (p < 0.001). The inspiratory volume calculated during the period when EAdi increased to its peak did not change from 276 +/- 147 to 277 +/- 162 ml, p = 0.976. Respiratory rate decreased from 23.9 (+/- 7) to 21.3 (+/- 7) breaths/min (p = 0.015). EAdi and Pdi decreased proportionally by adding PS (r = 0.84 and r = 0.90, for mean and peak values, respectively). Mean and peak EAdi decreased (p < 0.001) by 33 +/- 21% (mean +/- SD) and 37 +/- 23% with the addition of 10 cm H(2)O of PS, similar to the decrease in the mean and peak Pdi (p < 0.001) observed (34 +/- 36 and 35 +/- 23%). We also found that ventilator assist continued during the diaphragm deactivation period, a phenomenon that was further exaggerated at higher PS levels. We conclude that EAdi is a valid measurement of neural drive to the diaphragm in acute respiratory failure.

The use of esophageal recordings of the diaphragm electromyogram (EMG) signal strength to evaluate diaphragm activation during voluntary contractions in humans has recently been criticized because of a possible artifact created by changes in lung volume. Therefore, the first aim of this study was to evaluate whether there is an artifactual influence of lung volume on the strength of the diaphragm EMG during voluntary contractions. The second aim was to measure the required changes in activation for changes in lung volume at a given tension, i.e., the volume-activation relationship of the diaphragm. Healthy subjects (n = 6) performed contractions of the diaphragm at different transdiaphragmatic pressure (Pdi) targets (range 20-160 cmH2O) while maintaining chest wall configuration constant at different lung volumes. The diaphragm EMG was recorded with a multiple-array esophageal electrode, with control of signal contamination and electrode positioning. The effects of lung volume on the EMG were studied by comparing the crural diaphragm EMG root mean square (RMS), an index of crural diaphragm activation, with an index of global diaphragm activation obtained by normalizing Pdi to the maximum Pdi at the given muscle length (Pdi/Pdimax@L) at the different lung volumes. We observed a direct relationship between RMS and Pdi/Pdimax@L independent of diaphragm length. The volume-activation relationship of the diaphragm was equally affected by changes in lung volume as the volume-Pdi relationship (60% change from functional residual capacity to total lung capacity). We conclude that the RMS of the diaphragm EMG is not artifactually influenced by lung volume and can be used as a reliable index of diaphragm activation. The volume-activation relationship can be used to infer changes in the length-tension relationship of the diaphragm at submaximal activation/contraction levels.

SUMMARY Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids (SILACs) and cultured cells, we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared with quiescent cell counterparts. Bioinformatic analysis reveals that 44 of these proteins participate in hemostasis, a process not previously linked with cellular senescence. We validated the expression of some of these SASP factors in cultured cells and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, selective removal of senescent cells using transgenic p163MR mice showed that clearing senescent cells attenuates the increased clotting caused by doxorubicin. Our study provides an in-depth, unbiased analysis of the SASP and unveils a function for cellular senescence in hemostasis.

BackgroundExcessive respiratory muscle effort during mechanical ventilation may cause patient self-inflicted lung injury and load-induced diaphragm myotrauma, but there are no non-invasive methods to reliably detect elevated transpulmonary driving pressure and elevated respiratory muscle effort during assisted ventilation. We hypothesized that the swing in airway pressure generated by respiratory muscle effort under assisted ventilation when the airway is briefly occluded (ΔPocc) could be used as a highly feasible non-invasive technique to screen for these conditions.MethodsRespiratory muscle pressure (Pmus), dynamic transpulmonary driving pressure (ΔPL,dyn, the difference between peak and end-expiratory transpulmonary pressure), and ΔPocc were measured daily in mechanically ventilated patients in two ICUs in Toronto, Canada. A conversion factor to predict ΔPL,dyn and Pmus from ΔPocc was derived and validated using cross-validation. External validity was assessed in an independent cohort (Nanjing, China).ResultsFifty-two daily recordings were collected in 16 patients. In this sample, Pmus and ΔPL were frequently excessively high: Pmus exceeded 10 cm H2O on 84% of study days and ΔPL,dyn exceeded 15 cm H2O on 53% of study days. ΔPocc measurements accurately detected Pmus > 10 cm H2O (AUROC 0.92, 95% CI 0.83–0.97) and ΔPL,dyn > 15 cm H2O (AUROC 0.93, 95% CI 0.86–0.99). In the external validation cohort (n = 12), estimating Pmus and ΔPL,dyn from ΔPocc measurements detected excessively high Pmus and ΔPL,dyn with similar accuracy (AUROC ≥ 0.94).ConclusionsMeasuring ΔPocc enables accurate non-invasive detection of elevated respiratory muscle pressure and transpulmonary driving pressure. Excessive respiratory effort and transpulmonary driving pressure may be frequent in spontaneously breathing ventilated patients.

IntroductionBased on the hypothesis that failure of weaning from mechanical ventilation is caused by respiratory demand exceeding the capacity of the respiratory muscles, we evaluated whether extubation failure could be characterized by increased respiratory drive and impaired efficiency to generate inspiratory pressure and ventilation.MethodsAirway pressure, flow, volume, breathing frequency, and diaphragm electrical activity were measured in a heterogeneous group of patients deemed ready for a spontaneous breathing trial. Efficiency to convert neuromuscular activity into inspiratory pressure was calculated as the ratio of negative airway pressure and diaphragm electrical activity during an inspiratory occlusion. Efficiency to convert neuromuscular activity into volume was calculated as the ratio of the tidal volume to diaphragm electrical activity. All variables were obtained during a 30-minute spontaneous breathing trial on continuous positive airway pressure (CPAP) of 5 cm H2O and compared between patients for whom extubation succeeded with those for whom either the spontaneous breathing trial failed or for those who passed, but then the extubation failed.ResultsOf 52 patients enrolled in the study, 35 (67.3%) were successfully extubated, and 17 (32.7%) were not. Patients for whom it failed had higher diaphragm electrical activity (48%; P < 0.001) and a lower efficiency to convert neuromuscular activity into inspiratory pressure and tidal volume (40% (P < 0.001) and 53% (P < 0.001)), respectively. Neuroventilatory efficiency demonstrated the greatest predictability for weaning success.ConclusionsThis study shows that a mixed group of critically ill patients for whom weaning fails have increased neural respiratory drive and impaired ability to convert neuromuscular activity into tidal ventilation, in part because of diaphragm weakness.Trial RegistrationClinicaltrials.gov identifier NCT01065428. ©2012 Liu et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

IntroductionThe aim of this study was to validate an automated, objective and standardized algorithm for quantifying and displaying patient-ventilator interaction.MethodsUsing a new method to detect patient-ventilator synchrony, the present study re-analyzed previously acquired and published data from 24 mechanically ventilated adult patients (Colombo et al., Crit Care Med. 2011 Nov;39(11):2452–7). Patient-ventilator interactions were evaluated by comparing ventilator pressure and diaphragm electrical activity (EAdi) waveforms, recorded during pressure support ventilation. The EAdi and ventilator pressure waveforms were analyzed for their timings (manually and automatically determined), and the error between the two waveforms was quantified. A new index of patient-ventilator interaction (NeuroSync index), which is standardized and automated, was validated and compared to manual analysis and previously published indices of asynchrony.ResultsThe comparison of manual and automated detection methods produced high test-retest and inter-rater reliability (Intraclass correlation coefficient = 0.95). The NeuroSync index increased the sensitivity of detecting dyssynchronies, compared to previously published indices, which were found to only detect asynchronies.ConclusionThe present study introduces an automated method and the NeuroSync index to determine patient-ventilator interaction with a more sensitive analysis method than those previously described. A dashboard-style of graphical display allows a rapid overview of patient-ventilator interaction and breathing pattern at the bedside.