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.

Using injury cost functions from a predictive single-compartment model to assess the severity of mechanical ventilator-induced lung injuries.

Identifying safe ventilation patterns for patients with acute respiratory distress syndrome remains challenging because of the delicate balance between gas exchange and selection of ventilator settings to prevent further ventilator-induced lung injury (VILI). Accordingly, this work seeks to link ventilator settings to graded levels of VILI to identify injury cost functions that predict injury by using a computational model to process pressures and flows measured at the airway opening. Pressure-volume loops were acquired over the course of ~2 h of mechanical ventilation in four different groups of BALB/c mice. A cohort of these animals were subjected to an injurious bronchoalveolar lavage before ventilation. The data were analyzed with a single-compartment model that predicts recruitment/derecruitment and tissue distension at each time step in measured pressure-volume loops. We compared several injury cost functions to markers of VILI-induced blood-gas barrier disruption. Of the cost functions considered, we conclude that mechanical power dissipation and strain heterogeneity are the best at distinguishing between graded levels of injury and are good candidates for forecasting the development of VILI. NEW & NOTEWORTHY This work uses a predictive single-compartment model and injury cost functions to assess graded levels of mechanical ventilator-induced lung injury. The most promising measures include strain heterogeneity and mechanical power dissipation.

Linking Ventilator Injury-Induced Leak across the Blood-Gas Barrier to Derangements in Murine Lung Function.

Mechanical ventilation is vital to the management of acute respiratory distress syndrome, but it frequently leads to ventilator-induced lung injury (VILI). Understanding the pathophysiological processes involved in the development of VILI is an essential prerequisite for improving lung-protective ventilation strategies. The goal of this study was to relate the amount and nature of material accumulated in the airspaces to biomarkers of injury and the derecruitment behavior of the lung in VILI. Forty-nine BALB/c mice were mechanically ventilated with combinations of tidal volume and end-expiratory pressures to produce varying degrees of overdistension and atelectasis while lung function was periodically assessed. Total protein, serum protein, and E-Cadherin levels were measured in bronchoalveolar lavage fluid (BALF). Tissue injury was assessed by histological scoring. We found that both high tidal volume and zero positive end-expiratory pressure were necessary to produce significant VILI. Increased BALF protein content was correlated with increased lung derecruitability, elevated peak pressures, and histological evidence of tissue injury. Blood derived molecules were present in the BALF in proportion to histological injury scores and epithelial injury, reflected by E-Cadherin levels in BALF. We conclude that repetitive recruitment is an important factor in the pathogenesis of VILI that exacerbates injury associated with tidal overdistension. Furthermore, the dynamic mechanical behavior of the injured lung provides a means to assess both the degree of tissue injury and the nature and amount of blood-derived fluid and proteins that accumulate in the airspaces.

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.

Localized delivery of doxorubicin in vivo from polymer-modified thermosensitive liposomes with MR-guided focused ultrasound-mediated heating.

Thermosensitive liposomes have emerged as a viable strategy for localized delivery and triggered release of chemotherapy. MR-guided focused ultrasound (MRgFUS) has the capability of heating tumors in a controlled manner, and when combined with thermosensitive liposomes can potentially reduce tumor burden in vivo. However, the impact of this drug delivery strategy has rarely been investigated. We have developed a unique liposome formulation modified with p(NIPAAm-co-PAA), a polymer that confers sensitivity to both temperature and pH. These polymer-modified thermosensitive liposomes (PTSL) demonstrated sensitivity to focused ultrasound, and required lower thermal doses and were more cytotoxic than traditional formulations in vitro. A set of acoustic parameters characterizing optimal release from PTSL in vitro was applied in the design of a combined MRgFUS/PTSL delivery platform. This platform more effectively reduced tumor burden in vivo when compared to free drug and traditional formulations. Histological analysis indicated greater tumor penetration, more extensive ECM remodeling, and greater cell destruction in tumors administered PTSL, correlating with improved response to the therapy.

Effect of PEG molecular weight on stability, T2 contrast, cytotoxicity, and cellular uptake of superparamagnetic iron oxide nanoparticles (SPIONs).

Superparamagnetic iron oxide nanoparticles (SPIONs) are currently unavailable as MRI contrast agents for detecting atherosclerosis in the clinical setting because of either low signal enhancement or safety concerns. Therefore, a new generation of SPIONs with increased circulation time, enhanced image contrast, and less cytotoxicity is essential. In this study, monodisperse SPIONs were synthesized and coated with polyethylene glycol (PEG) of varying molecular weights. The resulting PEGylated SPIONs were characterized, and their interactions with vascular smooth muscle cells (VSMCs) were examined. SPIONs were tested at different concentrations (100 and 500 ppm Fe) for stability, T2 contrast, cytotoxicity, and cellular uptake to determine an optimal formulation for in vivo use. We found that at 100 ppm Fe, the PEG 2K SPIONs showed adequate stability and magnetic contrast, and exhibited the least cytotoxicity and nonspecific cellular uptake. An increase in cell viability was observed when the SPION-treated cells were washed with PBS after 1h incubation compared to 5 and 24h incubation without washing. Our investigation provides insight into the potential safe application of SPIONs in the clinic.