Unique Heterogeneous Topological Pattern of the Metabolic Landscape in Rabbit Fetal Lungs following Tracheal Occlusion

Abstract

Introduction: Fetal tracheal occlusion (TO) is currently an experimental approach to drive accelerated lung growth. It is stimulated by mechanotransduction that results in increased cellular proliferation and growth. However, it is currently unknown how TO affects the metabolic landscape of fetal lungs. Materials and Methods: TO or sham was performed on fetal rabbits at 26 days followed by lung harvest on day 30. Mass spectrometry was performed to evaluate global metabolic changes. Fluorescence lifetime intensity microscopy (FLIM) was performed to estimate local free/bound NADH relative ratio as an indicator of aerobic glycolysis versus oxidative phosphorylation (glycolysis/OXPHOS). Results: TO results in a metabolic shift from tricarboxylic acid cycle towards glycolysis. FLIM reveals uniform structures in control lungs characterized by similar ratios of free/bound NADH indicating a homogenous topological pattern. Similar uniform structures are observed in shams with some variability in the glycolysis/OXPHOS ratio. In contrast, lungs following TO demonstrate different types of unique distinct topological zones: one with enlarged alveoli and a shift towards glycolysis; the other maintains balance between glycolysis/OXPHOS similar to control lungs. Conclusion: We demonstrate for the first time a unique variable topological pattern of metabolism in fetal lungs following TO with a wide variation of metabolism between zones.

Neuronal Lipoprotein Lipase Deficiency Alters Neuronal Function and Hepatic Metabolism

“…The phasor transformation and the data analysis were carried out using Global SimFCS software (Laboratory for Fluorescence Dynamics (LFD), University of California, Irvine) as described previously [ 59 ]. The number of pixels covered with lifetimes for free and bound reduced forms of NADH and FAD were calculated in SimFCS (LFD), and the values were normalized to the total number of pixels detected as previously described [ 60 , 61 ].…”

Neuronal Lipoprotein Lipase Deficiency alters Neuronal Function and Hepatic Metabolism

“…The phasor transformation and data analysis were carried out using Global SimFCS software (Laboratory for Fluorescence Dynamics (LFD), University of California, Irvine) as described previously (25). The number of pixels covered with lifetimes for free and bound reduced form of NADH and FAD were calculated in SimFCS (LFD) and the values were normalized to the total number of pixels detected as previously described (26,27).…”

Heterogeneous Response in Rabbit Fetal Diaphragmatic Hernia Lungs After Tracheal Occlusion

“…Sample preparation, data acquisition, and analysis were performed as previously described and published by our group. 19,28 Data are graphically presented as heat maps relative to the corresponding median control value.…”

“…FLIM was performed to detect local metabolic changes in 20 different areas in fresh left and right lungs using a Zeiss 780 laser scanning confocal/multiphoton excitation fluorescence microscope with a 34-Channel GaAsP QUASAR Detection Unit and nondescanned detectors for 2-photon fluorescence (Zeiss, Thornwood, NY) equipped with an ISS A320 FastFLIM box and a titanium:sapphire Chameleon Ultra II (Coherent, Santa Clara, CA) as previously described. 19,28 For the acquisition of FLIM images, fluorescence for reduced form of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) was detected simultaneously by two photon-counting photomultiplier tube detectors (H7422p-40; Hamamatsu.) Images of the lung were obtained with Vista Vision software by ISS in 256 Â 256 format with a pixel dwell time of 6.3 ms/pixel and averaging over 30 frames.…”

“…1,30,31 TO has been shown to induce an accelerated lung growth in controls and CDH cases. 19,28,[31][32][33] Hence, an augmented total LBWR (TLBWR) is expected after TO. Consistently, the left CDH fetuses demonstrated a statistically significant decrease in TLBWR compared with control animals (0.012 AE 0.001 and 0.022 AE 0.001, respectively, P < 0.0001.)…”