A Comparative Study of Ex-Vivo Murine Pulmonary Mechanics Under Positive- and Negative-Pressure Ventilation.

Increased ventilator use during the COVID-19 pandemic resurrected persistent questions regarding mechanical ventilation including the difference between physiological and artificial breathing induced by ventilators (i.e., positive- versus negative-pressure ventilation, PPV vs NPV). To address this controversy, we compare murine specimens subjected to PPV and NPV in ex vivo quasi-static loading and quantify pulmonary mechanics via measures of quasi-static and dynamic compliances, transpulmonary pressure, and energetics when varying inflation frequency and volume. Each investigated mechanical parameter yields instance(s) of significant variability between ventilation modes. Most notably, inflation compliance, percent relaxation, and peak pressure are found to be consistently dependent on the ventilation mode. Maximum inflation volume and frequency note varied dependencies contingent on the ventilation mode. Contradictory to limited previous clinical investigations of oxygenation and end-inspiratory measures, the mechanics-focused comprehensive findings presented here indicate lung properties are dependent on loading mode, and importantly, these dependencies differ between smaller versus larger mammalian species despite identical custom-designed PPV/NPV ventilator usage. Results indicate that past contradictory findings regarding ventilation mode comparisons in the field may be linked to the chosen animal model. Understanding the differing fundamental mechanics between PPV and NPV may provide insights for improving ventilation strategies and design to prevent associated lung injuries.

Electrostatic properties of confluent Caco-2 cell layer correlates to their microvilli growth and determines underlying transcellular flow.

Recently, Rajapaksa et al. (2010) showed that the rate of uptake of potential vaccine delivery nanoparticles in the mucosal layer is a function of the electrostatic properties of the corresponding solvent. This fundamentally implies that the dominant driving forces that may be capitalized on for mucosal vaccine strategies are electrostatic in nature. We hypothesize that the driving force normal to the cell (in the direction from apical to basolateral across the cell) is of particular importance. In addition, it has been theoretically shown that the electrostatic properties of mucosal cells are directly related to their development of brush border. Here we correlate the development of brush border on a human mucosal epithelial model (Caco-2) cultured in DMEM on 3.0 µm pore sized polycarbonate membranes to their corresponding electrostatic properties characterized by measuring their normal zeta potential. Properties of normal streaming potential, hydraulic permeability, and brush border development (as determined by size and number) were monitored for 2, 6, and 16 days (after cells were confluent). Human endothelial cells (HECs), which lack brush border, were used as the control. Our results demonstrate that normal zeta potential of Caco-2 cells significantly changed from -5.7 ± 0.11 mV to -3.4 ± 0.11 mV for a period between 2 and 16 days, respectively. The zeta potential of the control cell line, HECs, stayed constant (statistically not different, P > 0.05) for the duration of the experiments. Our results show that the calculated increase in surface area of the Caco-2 cells with microvilli from 6 to 16 days was directly proportional to the corresponding measured zeta potential difference. These results imply that microvilli alter the electrostatic local environment around Caco-2 cells and, hence, enhance the normal electrostatic selective transport of solute across the mucosal barrier.

Novel in situ normal streaming potential device for characterizing electrostatic properties of confluent cells.

The characteristics of transport across confluent cell monolayers may often be attributed to its electrostatic properties. While tangential streaming potential is often used to quantify these electrostatic properties, this method is not effective for transport normal to the apical cell surface where the charge properties along the basolateral sides may be important (i.e., confluent cells with leaky tight junctions). In addition, even when cells have a uniform charge distribution, the shear stress generated by the conventional tangential flow device may dislodge cells from their confluent state. Here we introduce a novel streaming potential measurement device to characterize the normal electrostatic properties of confluent cells. The streaming potential device encompasses a 24 mm cell-seeded Transwell(®) with two AgCl electrodes on either side of the cell-seeded Transwell. Phosphate buffered saline is pressurized transversal to the Transwell and the resultant pressure gradient induces a potential difference. Confluent monolayers of HEK and EA926 cells are used as examples. The corresponding zeta potential of the cell-membrane configuration is calculated using the Helmholtz-Smoluchowski equation and the zeta potential of the confluent cell layer is deconvolved from the overall measurements. For these test models, the zeta potential is consistent with that determined using a commercial dispersed-cell device. This novel streaming potential device provides a simple, easy, and cost-effective methodology to determine the normal zeta potential of confluent cells cultured on Transwell systems while keeping the cells intact. Furthermore, its versatility allows periodic measurements of properties of the same cell culture during transient studies.