A novel method for pulmonary research: Assessment of bioenergetic function at the air–liquid interface

AbstractAir–liquid interface cell culture is an organotypic model for study of differentiated functional airway epithelium in vitro. Dysregulation of cellular energy metabolism and mitochondrial function have been suggested to contribute to airway diseases. However, there is currently no established method to determine oxygen consumption and glycolysis in airway epithelium in air–liquid interface. In order to study metabolism in differentiated airway epithelial cells, we engineered an insert for the Seahorse XF24 Analyzer that enabled the measure of respiration by oxygen consumption rate (OCR) and glycolysis by extracellular acidification rate (ECAR). Oxidative metabolism and glycolysis in airway epithelial cells cultured on the inserts were successfully measured. The inserts did not affect the measures of OCR or ECAR. Cells under media with apical and basolateral feeding had less oxidative metabolism as compared to cells on the inserts at air-interface with basolateral feeding. The design of inserts that can be used in the measure of bioenergetics in small numbers of cells in an organotypic state may be useful for evaluation of new drugs and metabolic mechanisms that underlie airway diseases

A novel method for pulmonary research: Assessment of bioenergetic function at the air–liquid interface

Air–liquid interface cell culture is an organotypic model for study of differentiated functional airway epithelium in vitro. Dysregulation of cellular energy metabolism and mitochondrial function have been suggested to contribute to airway diseases. However, there is currently no established method to determine oxygen consumption and glycolysis in airway epithelium in air–liquid interface. In order to study metabolism in differentiated airway epithelial cells, we engineered an insert for the Seahorse XF24 Analyzer that enabled the measure of respiration by oxygen consumption rate (OCR) and glycolysis by extracellular acidification rate (ECAR). Oxidative metabolism and glycolysis in airway epithelial cells cultured on the inserts were successfully measured. The inserts did not affect the measures of OCR or ECAR. Cells under media with apical and basolateral feeding had less oxidative metabolism as compared to cells on the inserts at air-interface with basolateral feeding. The design of inserts that can be used in the measure of bioenergetics in small numbers of cells in an organotypic state may be useful for evaluation of new drugs and metabolic mechanisms that underlie airway diseases

Mapping of vascular leakage in severe bTBI and naïve controls.

<p>For quantitative mapping of EB leakage, the entire brain was sliced into seven 2 mm thick sections. The brain sections were imaged at 24 h post-injury. <b>A)</b> Brain slices (left columns) represent optical images of brain tissue sections from severely injured group depicting an increase in EB dye leakage in the periventricular area (corpus callosum and hippocampus) and the occipital lobe. In control animals (without bTBI), EB dye leakage is seen in the choroid plexus and circumventricular organs, where there is no BBB. An actual photograph (right column) of the sliced brain sections (taken using HP Scanjet G4010) showing the different anatomical areas of interest. <b>B)</b> Mapping of EB dye leakage comparing naïve control and severe bTBI (120–125 psi) brains from six different anatomical areas: frontal lobe, corpus callosum, hippocampus, mid brain, and pons.</p

Effect of varying blast intensities on vascular leakage and BBB damage.

<p>Animals were exposed to mild (>20–50 psi), moderate (>50–90 psi) and severe (>90–130 psi) blast pressures. The damaged brains were harvested and imaged at 24 h post injury. <b>A)</b> A commensurate increase in the amount of EB dye leakage in brain tissue (in nanograms) and intensity of the shock waves used, i.e., mild (>20–50 psi), moderate (>50–90 psi) and severe (>90–130 psi). Data are shown as mean ± s.e.m., <i>n</i> = 4; <i>*p</i> ≤ 0.05. Statistical significance was calculated one-way ANOVA with Neuman-Keuls post-test. <b>B)</b> Representative images of immunofluorescently detected extravasated IgG in bTBI vs. naïve brains. Photo micrograph displays the increased IgG immunoreactivity in a region from the striatum of mild-bTBI brain as evidenced by enhanced fluorescent signal intensity. Absent leakage indicates integrity of the BBB as is evident in the naïve brain section which displays almost no fluorescent signal. Scale bar: 50 μm. <b>C)</b> Gross brain pathology following bTBI showed larger blood vessel diameters and hematomas (black arrows), more prominently in the frontal and occipital lobes (left side). Optical images of EB dye leakage shows prominent leakage in the brainstem area in bTBI animals.</p

Typical pressure-time profiles of the shock wave form.

<p><b>A)</b> Overview of the entire shock-wave profile. Pressure-time profiles of blast waves generated by different driver section configurations were typical to the Friedlander waveform of free-field blast waves. <b>B)</b> Detailed description of the peak overpressure, period of nonlinear decay, and positive pulse duration. A typical shock waveform lasted for 2–4 ms, depending on the driver length, and was composed of a steep shock front of positive overpressure, followed by a period of exponential decay with a negative vacuum phase.</p

Changes in ROS activity with blast conditions and with time following blast exposure:

<p>Animals exposed to different blast pressures and with time following blast exposure at mild condition were analyzed for ROS levels. The conversion of colorless CellROX™ dye to deep red fluorescence in the presence of ROS was captured using the Maestro Optical Imaging System. The total signal intensity from all brain slices together was measured, and the result was normalized per pixel to calculate ROS activity levels for each condition. Data are shown as mean ± s.e.m., <i>n</i> = 3–5 animals, *<i>p</i>≤ 0.05. Statistical significance was calculated using one-way ANOVA with Neuman-Keuls post-test.</p

Edema formation in severe bTBI and naïve controls.

<p>Animals were exposed to severe (120–125 psi) blast pressures and brains were harvested 3 h post-exposure. After excision, the damaged brains were dried for 48 h at 70°C and later weighed. Data are shown as mean ± s.e.m., <i>n</i> = 3,*<i>p</i> = 0.048. Statistical significance was calculated using one-tailed paired t-test with 95% confidence intervals.</p