The Evaluation of a Noninvasive Respiratory Volume Monitor in Mechanically Ventilated Neonates and Infants.

BACKGROUND: The respiratory volume monitor (RVM) (ExSpiron, Respiratory Motion Inc, Watertown, MA) uses thoracic impedance technology to noninvasively and continuously measure tidal volume (TV), respiratory rate (RR), and minute ventilation (MV). We aimed to validate the accuracy of the RVM to assess ventilation in neonates and infants by comparing it to spirometry. METHODS: We used the RVM and Respironics NM3 spirometer (Respironics NM3 Respiratory Profile Monitor, Philips Healthcare, Amsterdam, the Netherlands) to record simultaneous and continuous measurements of MV, TV, and RR. The RVM measurements, with and without external calibration, were compared to the Respironics NM3 spirometer using Bland-Altman analysis. The relative errors (Bland-Altman) between RVM and Respironics NM3 were calculated and used to compute individual patient bias, precision, and accuracy as the mean error, the standard deviation (SD) of the error, and the root mean square error. Bland-Altman limits of agreement (LoA) were computed, and equivalence tests were performed. RESULTS: Forty patients were studied to compare the RVM and Respironics NM3 measurements. The mean difference (ie, bias) for MV was 1.8% with 95% LoA, defined as mean ± 1.96 SD, in the range of -12.1% to 15.7%. Similarly, the mean difference (ie, bias) for TV and RR was 1.2% (95% LoA, -11.0% to 13.5%) and 0.6% (95% LoA, -3.7% to 5.0%), respectively. The mean measurement precision of the RVM relative to the Respironics NM3 for MV, TV, and RR was 10.8%, 8.9%, and 8.4%, respectively. The mean measurement accuracy for MV, TV, and RR across patients was 11.0%, 9.7%, and 7.1%, respectively. CONCLUSIONS: The data demonstrate that the RVM measures TV and MV in this cohort with an average relative error of 11% when using patient calibration and 16.9% without patient calibration. The average relative error of RR was 7.1%. The RVM provides accurate measurement of RR, TV, and MV in mechanically ventilated neonates and infants.

A High-Throughput System for Cyclic Stretching of Precision-Cut Lung Slices During Acute Cigarette Smoke Extract Exposure.

RATIONALE: Precision-cut lung slices (PCLSs) are a valuable tool in studying tissue responses to an acute exposure; however, cyclic stretching may be necessary to recapitulate physiologic, tidal breathing conditions. OBJECTIVES: To develop a multi-well stretcher and characterize the PCLS response following acute exposure to cigarette smoke extract (CSE). METHODS: A 12-well stretching device was designed, built, and calibrated. PCLS were obtained from male Sprague-Dawley rats (N = 10) and assigned to one of three groups: 0% (unstretched), 5% peak-to-peak amplitude (low-stretch), and 5% peak-to-peak amplitude superimposed on 10% static stretch (high-stretch). Lung slices were cyclically stretched for 12 h with or without CSE in the media. Levels of Interleukin-1ß (IL-1ß), matrix metalloproteinase (MMP)-1 and its tissue inhibitor (TIMP1), and membrane type-MMP (MT1-MMP) were assessed via western blot from tissue homogenate. RESULTS: The stretcher system produced nearly identical normal Lagrangian strains (E xx and E yy , p > 0.999) with negligible shear strain (E xy < 0.0005) and low intra-well variability 0.127 ± 0.073%. CSE dose response curve was well characterized by a four-parameter logistic model (R 2 = 0.893), yielding an IC50 value of 0.018 cig/mL. Cyclic stretching for 12 h did not decrease PCLS viability. Two-way ANOVA detected a significant interaction between CSE and stretch pattern for IL-1ß (p = 0.017), MMP-1, TIMP1, and MT1-MMP (p < 0.001). CONCLUSION: This platform is capable of high-throughput testing of an acute exposure under tightly-regulated, cyclic stretching conditions. We conclude that the acute mechano-inflammatory response to CSE exhibits complex, stretch-dependence in the PCLS.

Blood pressure-induced physiological strain variability modulates wall structure and function in aorta rings.

Vascular smooth muscle cells respond to mechanical stretch by reorganizing their cytoskeletal and contractile elements. Recently, we showed that contractile forces in rat aorta rings were maintained when the rings were exposed to 4 h of physiological variability in cycle-by-cycle strain, called variable stretch (VS), mimicking beat-to-beat blood pressure variability. Contractility, however, was reduced when the aorta was exposed to monotonous stretch (MS) with an amplitude equal to the mean peak strain of VS. OBJECTIVE: Here we reanalyzed the data to obtain wall stiffness as well as added new histologic and inhibitor studies to test the effects of VS on the extracellular matrix. MAIN RESULTS: The results demonstrate that while the stiffness of the aorta did not change during 4 h MS or VS, nonlinearity in mechanical behavior was slightly stronger following MS. The inhibitor studies also showed that mitochondrial energy production and cytoskeletal organization were involved in this fluctuation-driven mechanotransduction. Reorganization of ß-actin in the smooth muscle layer quantified from immunohistochemically labeled images correlated with contractile forces during contraction. Histologic analysis of wall structure provided evidence of reorganization of elastin and collagen fibers following MS but less so following VS. The results suggested that the loss of muscle contraction in MS was compensated by reorganization of fiber structure leading to similar wall stiffness as in VS. SIGNIFICANCE: We conclude that muscle tone modulated by variability in stretch plays a role in maintaining aortic wall structural and mechanical homeostasis with implications for vascular conditions characterized by a loss or an increase in blood pressure variability.

Low minute ventilation episodes during anesthesia recovery following intraperitoneal surgery as detected by a non-invasive respiratory volume monitor.

An electrical impedance-based noninvasive respiratory volume monitor (RVM) accurately reports minute volume, tidal volume and respiratory rate. Here we used the RVM to quantify the occurrence of and evaluate the ability of clinical factors to predict respiratory depression in the post-anesthesia care unit (PACU). RVM generated respiratory data were collected from spontaneously breathing patients following intraperitoneal surgeries under general anesthesia admitted to the PACU. Respiratory depression was defined as low minute ventilation episode (LMVe, < 40% predicted minute ventilation for at least 2 min). We evaluated for associations between clinical variables including minute ventilation prior to opioid administration and LMVe following the first PACU administration of opioid. Also assessed was a low respiratory rate (< 8 breaths per minute) as a proxy for LMVe. Of 107 patients, 38 (36%) had LMVe. Affected patients had greater intraoperative opioid dose, P = 0.05. PACU opioids were administered to 45 (42.1%) subjects, of which 27 (25.2%) had LMVe (P = 0.42) within 30 min following opioid. Pre-opioid minute ventilation < 70% of predicted normal value was associated with LMVe, P < 0.01, (sensitivity = 100%, specificity = 81%).Low respiratory rate was a poor predictor of LMVe (sensitivity = 11.8%). Other clinical variables (e.g., obstructive sleep apnea) were not found to be predictors of LMVe. Using RVM we identified that mild, clinically nondetectable, respiratory depression prior to opioid administration in the PACU was associated with the development of substantial subsequent respiratory depression during the PACU stay.

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors.

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging.

A microfluidic chamber-based approach to map the shear moduli of vascular cells and other soft materials.

There is growing interest in quantifying vascular cell and tissue stiffness. Most measurement approaches, however, are incapable of assessing stiffness in the presence of physiological flows. We developed a microfluidic approach which allows measurement of shear modulus (G) during flow. The design included a chamber with glass windows allowing imaging with upright or inverted microscopes. Flow was controlled gravitationally to push culture media through the chamber. Fluorescent beads were conjugated to the sample surface and imaged before and during flow. Bead displacements were calculated from images and G was computed as the ratio of imposed shear stress to measured shear strain. Fluid-structure simulations showed that shear stress on the surface did not depend on sample stiffness. Our approach was verified by measuring the moduli of polyacrylamide gels of known stiffness. In human pulmonary microvascular endothelial cells, G was 20.4 ± 12 Pa and decreased by 20% and 22% with increasing shear stress and inhibition of non-muscle myosin II motors, respectively. The G showed a larger intra- than inter-cellular variability and it was mostly determined by the cytosol. Our shear modulus microscopy can thus map the spatial distribution of G of soft materials including gels, cells and tissues while allowing the visualization of microscopic structures such as the cytoskeleleton.

Regulatory Roles of Fluctuation-Driven Mechanotransduction in Cell Function.

Cells in the body are exposed to irregular mechanical stimuli. Here, we review the so-called fluctuation-driven mechanotransduction in which stresses stretching cells vary on a cycle-by-cycle basis. We argue that such mechanotransduction is an emergent network phenomenon and offer several potential mechanisms of how it regulates cell function. Several examples from the vasculature, the lung, and tissue engineering are discussed. We conclude with a list of important open questions.

Homeostatic maintenance via degradation and repair of elastic fibers under tension.

Cellular maintenance of the extracellular matrix requires an effective regulation that balances enzymatic degradation with the repair of collagen fibrils and fibers. Here, we investigate the long-term maintenance of elastic fibers under tension combined with diffusion of general degradative and regenerative particles associated with digestion and repair processes. Computational results show that homeostatic fiber stiffness can be achieved by assuming that cells periodically probe fiber stiffness to adjust the production and release of degradative and regenerative particles. However, this mechanism is unable to maintain a homogeneous fiber. To account for axial homogeneity, we introduce a robust control mechanism that is locally governed by how the binding affinity of particles is modulated by mechanical forces applied to the ends of the fiber. This model predicts diameter variations along the fiber that are in agreement with the axial distribution of collagen fibril diameters obtained from scanning electron microscopic images of normal rat thoracic aorta. The model predictions match the experiments only when the applied force on the fiber is in the range where the variance of local stiffness along the fiber takes a minimum value. Our model thus predicts that the biophysical properties of the fibers play an important role in the long-term regulatory maintenance of these fibers.

Design of a Novel Equi-Biaxial Stretcher for Live Cellular and Subcellular Imaging.

Cells in the body experience various mechanical stimuli that are often essential to proper cell function. In order to study the effects of mechanical stretch on cell function, several devices have been built to deliver cyclic stretch to cells; however, they are generally not practical for live cell imaging. We introduce a novel device that allows for live cell imaging, using either an upright or inverted microscope, during the delivery of cyclic stretch, which can vary in amplitude and frequency. The device delivers equi-biaxial strain to cells seeded on an elastic membrane via indentation of the membrane. Membrane area strain was calibrated to indenter depth and the device showed repeatable and accurate delivery of strain at the scale of individual cells. At the whole cell level, changes in intracellular calcium were measured at different membrane area strains, and showed an amplitude-dependent response. At the subcellular level, the mitochondrial network was imaged at increasing membrane area strains to demonstrate that stretch can lead to mitochondrial fission in lung fibroblasts. The device is a useful tool for studying transient as well as long-term mechanotransduction as it allows for simultaneous stretching and imaging of live cells in the presence of various chemical stimuli.

Fluctuation-driven mechanotransduction regulates mitochondrial-network structure and function.

Cells can be exposed to irregular mechanical fluctuations, such as those arising from changes in blood pressure. Here, we report that ATP production, assessed through changes in mitochondrial membrane potential, is downregulated in vascular smooth muscle cells in culture exposed to monotonous stretch cycles when compared with cells exposed to a variable cyclic stretch that incorporates physiological levels of cycle-by-cycle variability in stretch amplitude. Variable stretch enhances ATP production by increasing the expression of ATP synthase's catalytic domain, cytochrome c oxidase and its tyrosine phosphorylation, mitofusins and PGC-1α. Such a fluctuation-driven mechanotransduction mechanism is mediated by motor proteins and by the enhancement of microtubule-, actin- and mitochondrial-network complexity. We also show that, in aorta rings isolated from rats, monotonous stretch downregulates-whereas variable stretch maintains-physiological vessel-wall contractility through mitochondrial ATP production. Our results have implications for ATP-dependent and mechanosensitive intracellular processes.

A novel device to stretch multiple tissue samples with variable patterns: application for mRNA regulation in tissue-engineered constructs.

A broad range of cells are subjected to irregular time varying mechanical stimuli within the body, particularly in the respiratory and circulatory systems. Mechanical stretch is an important factor in determining cell function; however, the effects of variable stretch remain unexplored. In order to investigate the effects of variable stretch, we designed, built and tested a uniaxial stretching device that can stretch three-dimensional tissue constructs while varying the strain amplitude from cycle to cycle. The device is the first to apply variable stretching signals to cells in tissues or three dimensional tissue constructs. Following device validation, we applied 20% uniaxial strain to Gelfoam samples seeded with neonatal rat lung fibroblasts with different levels of variability (0%, 25%, 50% and 75%). RT-PCR was then performed to measure the effects of variable stretch on key molecules involved in cell-matrix interactions including: collagen 1α, lysyl oxidase, α-actin, ß1 integrin, ß3 integrin, syndecan-4, and vascular endothelial growth factor-A. Adding variability to the stretching signal upregulated, downregulated or had no effect on mRNA production depending on the molecule and the amount of variability. In particular, syndecan-4 showed a statistically significant peak at 25% variability, suggesting that an optimal variability of strain may exist for production of this molecule. We conclude that cycle-by-cycle variability in strain influences the expression of molecules related to cell-matrix interactions and hence may be used to selectively tune the composition of tissue constructs.