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Heterogeneous Pulmonary Response After Tracheal Occlusion: Clues to Fetal Lung Growth
j o u r n a l o f s u r g i c a l r e s e a r c h j u l y 2 0 1 9 ( 2 3 9 ) 2 4 2 e2 5 2
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Heterogeneous Pulmonary Response After Tracheal Occlusion: Clues to Fetal Lung Growth Evgenia Dobrinskikh, PhD,a Saif I. Al-Juboori, PhD,b Uladzimir Shabeka, MD, PhD,b Julie A. Reisz, PhD,c Connie Zheng, BS,c and Ahmed I. Marwan, MDb,* a
Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado Division of Pediatric Surgery, Department of Surgery, University of Colorado School of Medicine, Aurora, Colorado c Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado b
article info
abstract
Article history:
Background: Understanding inconsistent clinical outcomes in infants with severe congen-
Received 27 October 2018
ital diaphragmatic hernia (CDH) after tracheal occlusion (TO) is a crucial step for advancing
Received in revised form
neonatal care. The objective of this study is to explore the heterogeneous airspace
15 January 2019
morphometry and the metabolic landscape changes in fetal lungs after TO.
Accepted 6 February 2019
Methods: Fetal lungs on days 1 and 4 after TO were examined using mass spectrometry
Available online xxx
ebased metabolomics, fluorescence lifetime imaging microscopy (FLIM), the number of airspaces, and tissue-to-airspace ratio (TAR).
Keywords:
Results: Two morphometric areas were identified in TO lungs compared with controls
Metabolic landscape
(more small airspaces at day 1 and a higher number of enlarged airspaces at day 4). Global
Heterogeneous metabolic zones
metabolomics analysis revealed a significant upregulation of glycolysis and a suppression
Tracheal occlusion
of the tricarboxylic acid cycle in day 4 TO lungs compared with day 1 TO lungs. In addition,
Pulmonary hypoplasia
there was a significant increase in polyamines involved in cell growth and proliferation.
Congenital diaphragmatic hernia
Locally, FLIM analysis on day 1 TO lungs demonstrated two types of heterogeneous
Fluorescence lifetime imaging
zonesdsimilar to control and with increased oxidative phosphorylation. FLIM on day 4 TO
microscopy
lungs demonstrated appearance of zones with enlarged airspaces and a metabolic shift toward glycolysis, accompanied by a decrease in the FLIM “lipid-surfactant” signal. Conclusions: In normal fetal lungs, we report a novel temporal pattern of varied morphometric and metabolic changes. Initially, there is formation of zones with small airspaces, followed by airspace enlargement over time. Metabolically day 1 TO lungs have zones with increased oxidative phosphorylation, whereas day 4 TO lungs have a shift toward glycolysis in the enlarged airspaces. Based on our observations, we speculate that the “best responders” to tracheal occlusion should have bigger lungs with small airspaces and normal surfactant production. ª 2019 Elsevier Inc. All rights reserved.
* Corresponding author. Division of Pediatric Surgery, Department of Surgery, University of Colorado School of Medicine, 12700 E 19th Avenue, MS 8618, Aurora, CO 80045. Tel.: þ303 724 4186; fax: þ303 724 6330. E-mail address: [email protected] (A.I. Marwan). 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.02.015
dobrinskikh et al heterogeneous lung response following to
Introduction Congenital diaphragmatic hernia (CDH) occurs in 1:2500-5000 births1,2 and results in pulmonary hypoplasia3 with different severity.4 Infants with the most severe left-sided CDH (observed-to-expected lung-to-head ratio of <25%) are currently offered an experimental interventiondtracheal occlusion (TO)dto accelerate lung growth (https://totaltrial.eu/). The technique of TO has undergone significant innovations from the initial report of “plug the lung until it grows (PLUG)” described by Hedrick et al. in 1994,5 to its current fetal endoscopic tracheal occlusion (FETO).6,7 In a few published series, reported survival rates after FETO were increased to 42%-48%;7,8 however, there have been inconsistent clinical outcomes after this intervention, the reasons of which are not fully understood. Tracheal occlusion is a classic example of how mechanosensitivity and mechanotransduction work in concert resulting in cellular proliferation and lung growth. It leverages the same mechanisms involved in pulmonary development that are dependent on the normal intrapulmonary pressure gradient generated by lung fluid production and the fixed resistance of the glottis9 accentuated by fetal breathing movements. Fetal lung development is a very complex process that follows a well-organized plan.10 Cells in the developing lungs are subjected to different forces, including tensile, compressive, hydrostatic, and fluid shear stress. The translation of local extrinsic mechanical events into fast and longstanding changes of cellular phenotype is dependent on the function of a complex cellular network of molecules and structures that are capable of sensing and responding to mechanical forces.10 Alveologenesis requires tightly regulated synchronization of multiple cell types.11,12 Thus, alveolar epithelial type 1 cells line the alveolus and form air-blood barrier, whereas alveolar epithelial type 2 cells produce and secrete surfactant, which reduces surface tension and preclude alveoli from collapsing after birth. Alveolar mesenchyme contains at least two types of fibroblasts: myofibroblasts, which produce elastin and can contract, and lipofibroblasts containing lipid droplets.13-17 Furthermore, the microvasculature of the lung contains the capillary network for gas exchange and the lymphatic network for removal of excess fluids. The interaction between mesenchymal and epithelial cells is important for cellular differentiation and function during alveolarization.18,19 Mechanical forces constitute significant stimuli for alveolarization.9,20 Surfactant production and release are stretch-dependent.21-23 Moreover, stretchdependent interactions of alveolar epithelial type 2 cells with fibroblasts can promote surfactant synthesis.22 Inhalation of amniotic fluid by prenatal breathing movements creates mechanical forces that are fundamental for the differentiation of alveolar epithelial type 1 cells.24 Also, mechanical forces can maintain mitotic spindle orientation in the airway cells and thus are important in airway morphogenesis.25 Along with other stimuli and forces, internal pressures due to fluid secretion across the pulmonary epithelium are fundamental for normal pulmonary development.9,26-28 Regulated fluid pressure has been shown to be essential for normal lung development by influencing epithelial folding and cellular proliferation.29 In addition, rapid cellular growth and proliferation require large amounts of individual building
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blocks including amino acids and nucleotides. Therefore, cells and tissues undergoing growth are expected to have metabolic changes commensurate to that dynamic process.30 It is not surprising that fetal pulmonary tissues preparing for an accelerated lung expansion after TO will shift their metabolism to glycolysis. In contrast to conventional glycolysis, actively growing cells shift the glycolytic flux toward lactate production through generation of large amounts of reduced nicotinamide adenine dinucleotide (NADH). Moreover, proliferating cells have active pentose phosphate shunt, which provides NADPH-reducing equivalents and five carbon units used to synthesize nucleotides.31-34 These processes allow for both adequate adenosine triphosphate (ATP) production and a surplus of carbon and NADH for growth.35 According to current paradigms, response of the fetal lungs to tracheal occlusion is viewed as uniform; however, we have previously suggested that the key principle in understanding changes in fetal lungs after TO is to view all concurrent changes from the perspective of biological heterogeneity.10,36 We have earlier demonstrated that after TO, the lungs will appear as a topologic mosaic characterized by a diverse metabolic landscape and parenchymal regions with variable structures in the newly formed lung tissue36; however, the natural history of changes in airspace morphometry and metabolic landscape after TO is currently unknown. The objective of this study is to examine the temporal pattern of changes in airspace morphometry and the metabolic landscape in normal fetal lungs after TO.
Material and methods Fetal rabbit tracheal occlusion model All animal procedures were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus (protocol #00434e6/2018). Time-dated pregnant New Zealand white rabbits were obtained at a gestational age of 17 to 19 d from Charles River Laboratory and housed in separate cages under standard laboratory conditions with free access to water and chow. Rabbits were allowed to acclimate for at least 5 d to compensate for the high altitude in Denver. On the day of surgery, each doe was premedicated with ketamine/xylazine (20-40 mg/kg and 5 mg/kg, respectively) and 7 mg medroxyprogesterone acetate (Depo-Provera) and were administered 300,000 units of penicillin G intramuscularly. Tracheal occlusion was performed on day 26 as described previously.36 Only two fetuses per doe were operated on, corresponding ovarianend position on opposite horns (TO and sham). When the ovarian-end fetus was smaller, a larger fetus toward the ovarian end and its corresponding position fetus on the opposite horn were selected for surgery. Fetal position was determined by gentle palpation followed by a 1-1.5 cm transverse hysterotomy on the antimesometrial border of the uterus. After delivery of the fetal head, a fetal midline neck incision was made. The trachea was dissected and separated
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from the esophagus, and a single 4-0 silk suture was passed around the trachea and gently tied in TO. A sham operation was performed on a fetus in a corresponding position on the opposite horn with exposure and dissection of the trachea, but no ligation. After surgery, fetuses were gently manipulated back into the uterine cavity. Three milliliters of warmed lactated Ringers solution was infused into the amniotic cavity to maintain amniotic fluid volume followed by closure of the hysterotomy using a running 4-0 silk suture. The uterus was placed back into the abdominal cavity and the abdomen closed in layers. All animals received bupivacaine 0.5% (12 mg/kg) and buprenorphine SR LAB (0.15 mg/kg) along the incision for postoperative analgesia in addition to 10 mL/kg of normal saline subcutaneously.
Harvest All fetuses were harvested by cesarean section on day 30. Does were subjected to the same premedication, anesthesia, and positioning. Three fetuses per doe (TO, sham, and unoperated pregnancy control) were delivered via a cesarean section. Immediate euthanasia was performed using approved methods. Successful TO was confirmed by naked eye examination, documenting larger lungs filling the chest cavity with an intact tracheal ligature and no apparent evidence of tracheal injury,
before tissue harvest. Lung tissue was immediately collected, flash-frozen in liquid nitrogen, and then stored in 80 C (for metabolomics and fluorescence lifetime imaging microscopy [FLIM]) or fixed in 4% paraformaldehyde for histological evaluation and morphometric analysis. Lung samples for morphometric analyses were not pressure-fixed as it has been previously demonstrated that morphometric indices can be compared in samples processed similarly, whether the fetal lungs are pressure- or immersion-fixed.2
Histological analysis Paraffin-embedded lungs were cut (5 mm sections) and stained with hematoxylin and eosin. Tissue sections were scanned using Aperio CS2 slide scanner (Leica Biosystems Inc, Buffalo Grove, IL) with 40 lens magnification; 20-30 random fields of view (FOVs) per animal (n ¼ 3, 3, 4; 5, 5, and 7 animals for control day 1, sham day 1, TO day 1; control day 4, sham day 4, and TO day 4, respectively, 2544 1295 pixels/image; image size was kept the same for all measurements) excluding conducting airways and major blood vessels were taken and analyzed in ImageJ (NIH, Bethesda, MD). Image acquisition and analysis were performed by different personnel (E.D. and S.I.A.-J.) with image analyses performed in a blinded fashion. Images were opened as RGB format and converted to 8-bit
Fig. 1 e TAR distribution analysis reveals at least two distinct regions in rabbit lung parenchyma after 1 d and 4 d following tracheal occlusion. (A) Hematoxylin and eosin representative images for lung tissue sections from control (top panels), sham (middle panels), and TO (bottom panels) samples at day 1 (left column) and day 4 (right column) after the surgeries are shown. In control tissues at day 1, there were a statistically significant smaller number of airspaces and tissue areas compared with day 4, which may correspond to normal lung growth. Lung tissues from sham samples 1 d after surgery showed airspaces that were similar to controls and others that were smaller (indicated by the arrows), suggesting possible induced branching as an acute response to lactate administration; however, at day 4, tissues from shams were indifferent from control samples, confirming an acute response at day 1. Lung tissues 1 d after TO demonstrated a further increase in the number of smaller airspaces (denoted by the arrows), indicating additional stimuli present in TO. With longer time (4 d) after surgery, there was an appearance of enlarged airspaces (denoted by arrowheads) in the TO samples. All TAR histograms for every condition were calculated and fitted to a gaussian fitting model. Raw data with the corresponding fits were plotted for combined histograms at day 1 (B) and day 4 (E) after surgery. Day 1 sham and TO histograms were further separated into two distributions: control-like TAR (C) and bigger TAR (smaller airspaces) (D) subsets. The day 4 sham histogram was similar to control and therefore was not separated (E-G). In contrast to day 1, the day 4 TO histogram was separated into control-like TAR (F) and smaller TAR (enlarged airspaces) (G) subsets. Student’s two-tailed t-test was used for statistical comparison. n [ 3-7 animals/group. Significantly different groups were denoted with **, P < 0.02. (Color version of figure is available online.)
dobrinskikh et al heterogeneous lung response following to
grayscale. Airspaces were thresholded using the corresponding background histogram and analyzed with Analyze Particles plugin tool. Particles greater than 500-pixel square were segmenteddcircularity range was 0-1, and holes were included. Areas of each individual particle were recovered. Consequently, tissue areas were calculated by subtracting the sum of total airspace areas from image size. The number of airspaces and tissue-to-airspace ratios (TARs) were calculated. TAR histograms were calculated and fitted to a least square Gaussian fitting model, and their means and standard deviations were extracted from the fits for statistical significance comparison purposes.
Mass spectrometryebased metabolomics analysis Global metabolomics analysis was performed on frozen lung samples from control, sham, and TO using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass spectrometer. Sample preparation, data acquisition, and analysis were performed as previously described.36 Data are graphically presented relative to the median control value at the respective time point.
Fluorescence lifetime imaging microscopy FLIM was performed to detect local metabolic changes in 15-20 different areas in a fresh lung lobe (which was kept the same for each animal) using a Zeiss 780 laser-scanning confocal/ multiphoton-excitation fluorescence microscope with a
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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). A dichroic filter (496 nm; Semrock Inc, Rochester, NY) was used to separate the fluorescence signal. The 2photon excitation was blocked by a 2-photon emission filter. For the acquisition of FLIM images, fluorescence was detected by a photon-counting photomultiplier tube detector (H7422p40; Hamamatsu Photonics, Hamamatsu, Japan). Images of the lung were obtained with the Vista Vision software by ISS in 256 256 format with a pixel dwell time of 6.3 ms/pixel and averaging over 30 frames. Calibration of the system was performed by measuring the known lifetime of the fluorophore fluorescein with a single exponential decay of 4.0 ns.37 The phasor transformation and data analysis were carried out using the Global SimFCS software (Laboratory for Fluorescence Dynamics, University of California, Irvine, CA) as described previously.38 The number of pixels covered with lifetimes for free and bound NADH, as well as “lipid-surfactant” signals, was calculated in SimFCS (Laboratory for Fluorescence Dynamics), and the values were normalized to the total number of pixels detected. Data are shown as a percentage of total in pie charts.
Statistical analysis Data were expressed as means SEM. Three-seven (n ¼ 3-7) animals/group were used. Two-sample t-test (two-tailed
Fig. 2 e Arginine metabolism is increased at day 4 after tracheal occlusion. A classical arginine metabolic pathway is accompanied by plots of metabolomics results where values are normalized to their respective day’s control group. n [ 4-7/ group. Significantly increased spermidine and spermine and decreased creatine and proline were found in TO samples 4 d after surgery. Increased spermidine and spermine levels indicate increased cell proliferation, whereas creatinine is possibly providing some required energy for cell growth. Decreased levels of fumarate and trend toward decreased levels of aspartate may indicate that arginine metabolism is shifting these intermediates toward polyamines production shunting them away from TCA cycle. Student’s t-test was used for statistical comparison between day 1 and day 4 TO groups. Significantly different metabolites are denoted with * where P < 0.05 and ** with P < 0.01.
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unpaired Student’s t-test) was performed for statistical analysis of histograms, using a 0.01 significance level and a twosided P-value. Two-sample t-test (two-tailed unpaired Student’s t-test, significance threshold P < 0.05) for metabolomics results was performed using the Prism 7.0 software (GraphPad, San Diego, CA). Statistical differences (P < 0.05) between groups for FLIM data were determined by one-way analysis of variance (Tukey correction) for multiple comparisons using the Prism 7.0 software (GraphPad).
Results Morphometric analysis reveals two unique distributions of distal airspaces in fetal rabbit lung tissues after TO To examine the natural history of parenchymal airspace morphometric changes in fetal lungs after TO, TARs were calculated at day 1 and day 4 after TO surgery and compared with their control and sham counterparts (Fig. 1). Images of hematoxylin and eosinestained lung tissue sections were used to calculate TARs for each condition, as described in the Methods Section. We found that the number of airspaces per FOV was significantly less in day 1 control samples than in day 4 control samples (83 2 at day 1 compared with 91 1 at day 4, P < 0.0005), likely corresponding to a normal lung growth pattern. At day 1, sham samples had some regions with smaller airspaces (Fig. 1A, middle left panel, denoted by arrows) than controls, whereas sham day 4 tissues were indifferent from controls (Fig. 1A, middle right panel), indicating
possible induced branching at day 1 due to an acute response to lactate administration. The number of smaller airspaces was higher in TO samples at day 1 than shams, suggesting an extra stimulation due to occlusion (Fig. 1A, bottom left panel, denoted by arrows). After 4 d of TO, enlarged airspaces were observed as shown in Figure 1A (bottom right panel, denoted by arrowheads). For each condition, a TAR histogram was calculated and fitted to a least-square Gaussian fitting model (Fig. 1B-G). Combined histograms for sham and TO samples at day 1 had a significant shift toward bigger TAR values (smaller airspaces), which was more pronounced in TO (Fig. 1B). To demonstrate the heterogeneous response to tracheal occlusion, we separated the corresponding sham and TO TAR histograms into two subdistributions: TAR values that are greater than the mean of TAR control plus one standard deviation (mean þ s) were assigned to the bigger TAR (smaller airspaces) (Fig. 1D) subdistribution, and the rest were considered as control-like TARs (Fig. 1C). These TAR subdistributions were statistically different from control and each other (Fig. 1D). On the other hand, combined histograms for TO at day 4 demonstrated a significant shift toward smaller TAR values (bigger airspaces), compared with control and sham (Fig. 1E). In a similar fashion, we separated TAR histogram values in TO samples into two subdistributions: TAR values that are smaller than the mean of TAR control minus one standard deviation (mean s) were allocated to the smaller TAR (enlarged airspaces) (Fig. 1G) subdistribution, and the remaining values were considered as similar to control (Fig. 1F). TO small TAR subdistribution was statistically different from control and sham TAR distributions (Fig. 1G).
Fig. 3 e Tricarboxylic acid (TCA) cycle is suppressed at day 4 after TO. In all graphs, metabolic data are shown relative to control. n [ 4-7/group. Metabolic analysis demonstrated a decline in the TCA cycle with a significant decrease in citrate and fumarate in day 4 compared with day 1 TO lungs. This suggests a high demand for energy and metabolite production and a shift to faster glycolysis. Student’s two-tailed t-test was used for statistical comparison between day 1 and day 4 TO groups. Significantly different metabolites are denoted with ** where P < 0.01.
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Fig. 4 e Glycolysis is upregulated in the fetal lungs at day 4 after TO. In all graphs, metabolic data are shown as relative to control. n [ 4-7/group. Glucose and hexose phosphate were significantly decreased in day 4 compared with day 1 TO lungs. This was accompanied by a significant shift toward lactate production at day 4 after TO compared with day 1. Student’s twotailed t-test was used for statistical comparison between day 1 and day 4 TO groups. Significantly different metabolites are denoted with * where P < 0.05.
Day 4 sham TAR distribution was not statistically different from that of day 4 control and thereby was not separated into the two subdistributions mentioned previously.
The global metabolic landscape after TO Metabolism impacts all cellular functions, including cell proliferation and differentiation required for lung development. Untargeted high-throughput metabolomics was performed using ultra high-pressure liquid chromatography coupled online to mass spectrometry (UHPLC-MS) and demonstrated a shift from mitochondrial metabolism to glycolysis 4 d after TO in comparison with sham and control, indicating high demands for energy in occluded lungs.
Upregulated arginine metabolism at day 4 after TO Arginine cycle metabolites play important roles in multiple pathways (a schematic presentation of a classical arginine metabolic pathway is depicted in Fig. 2); for example, creatine is necessary for ATP production,39 and ornithine is involved in cell proliferation, extracellular matrix (ECM) remodeling, and nitrogen excretion via the urea cycle. For cell proliferation processes, ornithine is converted to putrescine followed by spermidine and spermine.40 Conversion to proline is essential
for ECM proteins that are rich in proline and glycine production, such as collagens and elastin.41 We have found significantly increased levels of spermidine and spermine (Fig. 2) and a trend toward a decrease in proline and creatine in day 1 TO lungs compared with day 4 TO lungs. In addition, we have found statistically indifferent levels of aspartate and decreased fumarate in day 1 TO lungs compared with day 4 TO lungs (Fig. 2), which might indicate that arginine metabolism is shifting these intermediates toward polyamine production, channeling them away from the tricarboxylic acid (TCA) cycle.
TCA cycle suppression and glycolysis upregulation at day 4 after TO Both the TCA cycle (Fig. 3) and glycolysis (Fig. 4) play crucial roles in ATP production, but their speeds and efficiencies are different42 and metabolic flexibility is very important for adaptation to different microenvironmental conditions.43 Differentiated cells mostly use oxidative phosphorylation (OXPHOS) to efficiently generate necessary amounts of ATP,30,44 whereas rapidly proliferating cells and cells devoid of adequate oxygen shift to increased nutrient uptake and glycolysis.30 Metabolomics analysis revealed a decline in the TCA cycle ascertained by a significant decrease in citrate and fumarate in day 4 TO lungs compared with day 1 TO lungs (Fig. 3). In addition, glucose
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Fig. 5 e Mitochondrial transport and fatty acid metabolism. In all graphs, metabolic data are shown as relative to control. n [ 4-7/group. Caprylate (C 8:0) and pelargonate (C 9:0) were decreased at day 4 after TO. Most metabolites involved in the carnitine shuttle were increased at day 4 after TO compared with day 1. Student’s two-tailed t-test was used for statistical comparison between day 1 and day 4 TO groups. Significantly different metabolites are denoted with * where P < 0.05 and ** with P < 0.01.
and hexose phosphate levels were significantly decreased in day 4 TO lungs compared with day 1 TO lungs (Fig. 4). This was accompanied by a significant shift toward lactate production in day 4 TO lungs compared with day 1 TO lungs (Fig. 4), suggesting the use of ongoing glycolysis to meet the increased needs for energy production in occluded lungs.
Mitochondrial transport and fatty acids beta-oxidation are altered in TO lungs Fatty acid beta-oxidation occurs primarily in the mitochondria and is important for ATP and NADH generation.45 It also generates the TCA cycle metabolite acetyl coenzyme A (acetylCoA). To enter mitochondria, fatty acids attached to coenzyme A (acyl-CoA) are shuttled into the mitochondria as acylcarnitine intermediates46,47 (Fig. 5). Following translocase activity, acylcarnitines are converted back to their acyl-CoA forms, allowing beta-oxidation to occur. We found that the level of caprylate (C 8:0) was significantly decreased and pelargonate (C 9:0) had a trend to decrease at day 4 after TO (Fig. 5). Four days after TO, there was an observed increase in most metabolites involved in the carnitine shuttle compared with day 1 (Fig. 5).
The local metabolic landscape after tracheal occlusion Metabolic changes at day 1 after TO are consistent with an increase in oxidative phosphorylation FLIM was used to examine local metabolic changes in fetal lungs at day 1 and day 4 after TO. Although intensity images of
multiple random FOVs at day 1 were similar in all examined groups of animals (Fig. 6A, top panel), corresponding FLIM maps showed heterogeneous lifetime allocations in the TO group, compared with sham and control. Lifetime distribution in day 1 TO lungs revealed two distinct regionsdregions similar to control and regions with a visually increased lipid-surfactant signal (Fig. 6A, bottom panel). Quantification of percentages covered by specific lifetime signals and free/bound NADH ratios in control and sham animals demonstrated a nonstatistically significant decrease in the NADH lifetime percentages (Fig. 6B, 9 1% for free NADH and 58 2% for bound NADH in shams versus 11 2% for free NADH and 63 2% for bound NADH in controls) and the free/bound NADH ratio in shams compared with controls (Fig. 6C, 0.16 0.02 compared with 0.21 0.04, respectively). On the other hand, both free and bound NADH lifetime percentages (Fig. 6B, 7 1% for free NADH and 49 2% for bound NADH in TO versus 11 2% for free NADH and 63 2% for bound NADH in controls, P < 0.0001) and free/bound NADH ratio were significantly decreased in combined analysis of all images from day 1 TO lungs compared with controls (Fig. 6C, 0.11 0.01 versus 0.21 0.04, respectively, P ¼ 0.0017). In addition to decreased free/bound NADH ratio, indicating a shift toward OXPHOS,48 there was an increase in the percentage of pixels with lipidsurfactant signals (Fig. 6B, cyan and magenta sectors), suggesting a possible surfactant release, perhaps as a response to the initial mechanical stretch stimulus after TO surgery; however, after the separation of regions with dissimilar FLIM maps from the occluded fetal lungs, we demonstrated that a decrease in
dobrinskikh et al heterogeneous lung response following to
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Fig. 6 e Local metabolic landscape demonstrates the presence of at least two different regions after tracheal occlusion. (A) Representative intensity images (top row) with corresponding FLIM maps (bottom row) of control, sham, and TO lungs at day 1 after surgery are shown. Fetal lungs in all conditions demonstrated similar structures; however, the local metabolic landscape was heterogenous in lungs after TO. (B) Graphical representation of percentage covered by specific lifetime signals in controls and sham animals exhibited a nonstatistically significant decrease in the free/bound NADH ratio in shams compared with controls. Combined regions of TO lungs showed a significant decrease in the free/bound NADH ratio (C) and an increased lipidsurfactant signal, indicating a surfactant release and shift toward OXPHOS, probably as a response to initial mechanical stretch after TO surgery. Separation of zones based on their FLIM maps revealed presence of zones with similar to control signatures and a more pronounced significant decrease in the free/bound NADH ratio (D) with an increased lipid-surfactant signal. (E) Representative intensity images (top row) with corresponding FLIM maps (bottom row) of control, sham, and TO lungs at day 4 after surgery are shown. Control and sham lungs displayed similar structures with a homogeneous metabolic landscape. Conversely, TO demonstrated two distinct types of zones; one type of zones was structurally similar to both controls and shams and had a similar free/bound NADH ratio (H). The second type of zones in TO demonstrated evidence of airspaces enlargement and is characterized by a significant shift toward glycolysis and an decreased lipid-surfactant signal. (F) Graphical representation of percentage covered by specific lifetime signals in controls, shams, and combined regions of TO animals demonstrated a nonstatistically significant increase in the free/bound NADH ratio (G) in shams compared with controls. One-way analysis of variance (Tukey correction) was used for statistical comparison between the groups. n [ 4-7 animals/group. Statistical differences were determined as significant for P < 0.05. (Color version of figure is available online.)
free/bound NADH ratio (Fig. 6B, TO separated; Fig. 6D red shaded bar, TO OXPHOS [0.07 0.01]) was observed in the areas with a increased lipid-surfactant signal, whereas the remaining zones were similar to shams and controls (Fig. 6B, TO separated; Fig. 6D purple shaded bar, TO control [0.22 0.03]). In contrast to histological findings, local metabolic changes in day 1 sham lungs were not different from controls (Fig. 6A-C), indirectly confirming a possible acute transient effect secondary to intra-amniotic lactate instillation.
Enlarged airspaces 4 d after TO are associated with a shift toward glycolysis Similar to the histological changes, at day 4 after occlusion, we observed heterogeneous intensity images: images with controllike and images with enlarged parenchymal structures,
whereas sham and control intensity images were indifferent from each other (Fig. 6E, top panel). Corresponding FLIM maps revealed a unique increase in free NADH lifetime in regions with enlarged structures in TO lungs (Fig. 6E, bottom panel), indicating a shift toward glycolysis48 in these areas. Quantification of percentages covered by particular lifetime signals in control and sham animals demonstrated similar percentages for free and bound NADH lifetimes (Fig. 6F, 28 2% for free NADH and 62 1% for bound NADH in shams versus 21 1% for free NADH and 73 2% for bound NADH in controls) and a nonstatistically significant increase in the free/bound NADH ratio in shams compared with controls (Fig. 6G, 0.53 0.05 versus 0.35 0.08, respectively). Analysis of all images taken in animals after TO demonstrated a significant increase of free (and decrease of bound) NADH lifetime percentage(s) (Fig. 6F, 39
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Fig. 7 e Model of lung development after tracheal occlusion (TO). The five well-known stages for normal lung development, their major characterizing events, and corresponding rabbit gestational ages adapted from Surate Solaligue et al.52 are depicted. TO is introduced at E26, which leads to accumulation of fluid containing different stimuli, including growth factors and metabolites. Our morphometric findings (highlighted with blue font) showeddin addition to control-likedan appearance of smaller airspaces at day 1 after TO. This corresponded to greater TAR values, suggesting an increased branching, whereas 4 d after TO, there is an appearance of enlarged (highlighted with red font and arrow) airspaces resulted in reduced TAR values, presumably induced by increased fluid accumulation in these regions and control-like TAR values (highlighted with green font and arrows). The natural history of the local metabolic FLIM signals (highlighted with brown font) suggests that there is an increase in OXPHOS and “lipid-surfactant” signals in some areas at day 1 after the occlusion, relative to control; however, the “lipid-surfactant” FLIM signal is reduceddand accompanied by a significant metabolic shift toward glycolysisdin the enlarged airspaces at day 4 after TO, whereas control-like airspaces, at day 4, maintain both a normal “lipid-surfactant” FLIM signal and a balanced metabolism. Based on our observations and assumptions, we would anticipate that the “best responders” to tracheal occlusion should have bigger lungs with more control-like airspaces maintaining normal surfactant production. (Color version of figure is available online.)
2% for free NADH and 58 2% for bound NADH in TO versus 21 1% for free NADH and 73 2% for bound NADH in controls, P < 0.0001) and a significant free/bound NADH ratio increase in day 4 TO lungs compared with controls (Fig. 6G, 0.92 0.11 versus 0.35 0.08, respectively, P ¼ 0.0006). Moreover, there was a substantial decrease in the percentage of pixels with lipidsurfactant signals in all group of animals, compared with day 1 counterparts (Fig. 6F versus Fig. 6B), perhaps due to observed overall increased TAR values (i.e., smaller airspaces) for day 4 lungs compared with day 1 lungs (Fig. 1E versus Fig. 1B). The TO group had a further decrease in the lipid-surfactant signal, which agrees with the decrease in surfactant production reported previously.49,50 After separation of regions with dissimilar structures, from the occluded fetal lungs, we observed an increased free/bound NADH ratio and an almost absent lipid-surfactant signal in the areas with enlarged airspaces (Fig. 6F, TO separated; Fig. 6H red bar, TO glycolysis [1.45 0.19]), indicating a shift toward glycolysis possibly due to a higheenergy demand environment.
Discussion In this article, we describe the temporal pattern of the heterogeneous airspace morphometry and the metabolic
landscape changes in normal fetal lungs undergoing TO. Previously, we have proposed and demonstrated the heterogeneity concept of TO lung growth by virtue of the local metabolic landscape using FLIM on lung lobes at day 4.36 Here, we used morphometric measures to confirm our findings on the histological level, explored the natural history of the morphometric heterogeneity after TO, and described the global and local metabolic changes. Overall, the number of airspaces and tissue areas were less in day 1 control samples than day 4 samples, indicating normal lung growth. Our observation of an increase in smaller airspaces in day 1 sham samples may be attributed to an induced branching at day 1 due to an acute response to lactate administration upon hysterotomy closure. With regard to global changes in metabolism after TO, it is not surprising that by day 4, there was a shift away from TCA and toward glycolysis. This finding is in agreement with a “Warburg-like” metabolism being adopted by actively proliferating tissues to support growth30; however, a possible explanation for the early (day 1) local metabolic FLIM findings may be related to the stretch of type 2 cells in response to mechanical stimulation after occlusion. This correlates with the OXPHOS-driven metabolic landscape at day 1. Consistent with the anticipated stretch of type 2 cells, it is expected that there will be an increase in the surfactant signal as seen in our
dobrinskikh et al heterogeneous lung response following to
day 1 FLIM data; however, as occlusion is maintained for 4 d, cells will start to divide, and therefore a shift in the metabolic landscape toward glycolysis is needed. At day 4, the FLIM lipid-surfactant signal decreased, suggesting a decrease in surfactant production, which was previously shown in proliferating type 2 cells.51 Another explanation for the reduction in surfactant production, suggested by De Paepe et al., may be attributed to an accelerated terminal differentiation of type 2 to type 1 cells.49 One interesting observation with regard to the FLIM lipidsurfactant signal is the lower baseline FLIM signal in the day 4 controls compared with day 1 controls. This reduction can be explained by the expected lung growth from day 26 to day 30 (which will result in more tissue, i.e., more NADH FLIM signal compared with “lipid-surfactant” counterpart) and, therefore, the relative percentage decrease of the surfactant signal. Although we showed that changes in FLIM signals were observed in enlarged airspaces, it is currently unknown whether these FLIM lipid-surfactant signals are associated with changes in ECM proteins production to support alveolar wall. Furthermore, it will be important to examine the correlation between these changes and the appearance of heterogeneous histological and metabolic zones. Although these observations are fundamental in understanding the metabolic response of normal lungs to occlusion, it will be important to examine the metabolic landscape in fetuses who have undergone a surgical creation of a CDH followed by TO. These experiments are currently underway in our laboratory.
Conclusions In conclusion, in this article, we confirm our concept of formation of heterogeneous zones in occluded fetal lungs and describe the natural history of the morphometric and metabolic changes after TO (Fig. 7). Initially there is formation of zones with small airspaces, followed by airspace enlargement over time. Metabolically, day 1 TO lungs have zones with increased OXPHOS, whereas day 4 TO lungs have a shift toward glycolysis in the enlarged airspaces. Thus, we highlight the relationship between metabolic and morphometric changes to prime the pulmonary machinery for mechano-dependent lung growth.
Acknowledgment The authors thank the University of Colorado School of Medicine, the University of Colorado Veterinary services for their assistance with animal surgeries and postsurgical care, and the Morphology Core for their assistance with sample preparation and processing. Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core supported in part by NIH/ NCATS Colorado CTSI Grant Number UL1 TR001082. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. Authors’ contribution: E.D. conceptualized the study; performed the histological staining and imaging and FLIM imaging
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and analyses; analyzed and interpreted the data; and contributed to manuscript writing, editing, and critical review. S.I.A.-J. performed histological analysis, histograms fitting, and statistical comparisons and contributed to manuscript writing, editing, and critical review. U.S. and A.I.M. conceptualized the study, performed the animal surgeries, analyzed and interpreted the data, and contributed to manuscript writing, editing, and critical review. J.A.R. and C.Z. performed metabolomics and contributed to manuscript editing and critical review.
Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.
references
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35. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab. 2008;7:11e20. 36. Marwan AI, Shabeka U, Reisz JA, Zheng C, Serkova NJ, Dobrinskikh E. Unique heterogeneous topological pattern of the metabolic landscape in rabbit fetal lungs following tracheal occlusion. Fetal Diagn Ther. 2018. 37. Berezin MY, Achilefu S. Fluorescence lifetime measurements and biological imaging. Chem Rev. 2010;110:2641e2684. 38. Digman MA, Caiolfa VR, Zamai M, Gratton E. The phasor approach to fluorescence lifetime imaging analysis. Biophys J. 2008;94:L14eL16. 39. Abraham MR, Bottomley PA, Dimaano VL, et al. Creatine kinase adenosine triphosphate and phosphocreatine energy supply in a single kindred of patients with hypertrophic cardiomyopathy. Am J Cardiol. 2013;112:861e866. 40. Wei LH, Wu G, Morris Jr SM, Ignarro LJ. Elevated arginase I expression in rat aortic smooth muscle cells increases cell proliferation. Proc Natl Acad Sci U S A. 2001;98:9260e9264. 41. Li P, Wu G. Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth. Amino Acids. 2018;50:29e38. 42. Zheng J. Energy metabolism of cancer: glycolysis versus oxidative phosphorylation (Review). Oncol Lett. 2012;4:1151e1157. 43. Jose C, Bellance N, Rossignol R. Choosing between glycolysis and oxidative phosphorylation: a tumor’s dilemma? Biochim Biophys Acta. 2011;1807:552e561. 44. Khairallah M, Labarthe F, Bouchard B, Danialou G, Petrof BJ, Des Rosiers C. Profiling substrate fluxes in the isolated working mouse heart using 13C-labeled substrates: focusing on the origin and fate of pyruvate and citrate carbons. Am J Physiol Heart Circ Physiol. 2004;286:H1461eH1470. 45. Sebastian D, Guitart M, Garcia-Martinez C, et al. Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells. J Lipid Res. 2009;50:1789e1799. 46. Longo N, Frigeni M, Pasquali M. Carnitine transport and fatty acid oxidation. Biochim Biophys Acta. 2016;1863:2422e2435. 47. Liepinsh E, Makrecka-Kuka M, Kuka J, et al. Inhibition of Lcarnitine biosynthesis and transport by methyl-gammabutyrobetaine decreases fatty acid oxidation and protects against myocardial infarction. Br J Pharmacol. 2015;172:1319e1332. 48. Stringari C, Nourse JL, Flanagan LA, Gratton E. Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential. PLoS One. 2012;7:e48014. 49. De Paepe ME, Johnson BD, Papadakis K, Sueishi K, Luks FI. Temporal pattern of accelerated lung growth after tracheal occlusion in the fetal rabbit. Am J Pathol. 1998;152:179e190. 50. Flecknoe SJ, Wallace MJ, Cock ML, Harding R, Hooper SB. Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep. Am J Physiol Lung Cell Mol Physiol. 2003;285:L664eL670. 51. Smith LJ. The effect of type 2 cell mitosis on the surfactant system of injured mouse lungs. J Lab Clin Med. 1983;102:434e443. 52. Surate Solaligue DE, Rodriguez-Castillo JA, Ahlbrecht K, Morty RE. Recent advances in our understanding of the mechanisms of late lung development and bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol. 2017;313:L1101eL1153.
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Association for Academic Surgery
Heterogeneous Pulmonary Response After Tracheal Occlusion: Clues to Fetal Lung Growth Evgenia Dobrinskikh, PhD,a Saif I. Al-Juboori, PhD,b Uladzimir Shabeka, MD, PhD,b Julie A. Reisz, PhD,c Connie Zheng, BS,c and Ahmed I. Marwan, MDb,* a
Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado Division of Pediatric Surgery, Department of Surgery, University of Colorado School of Medicine, Aurora, Colorado c Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado b
article info
abstract
Article history:
Background: Understanding inconsistent clinical outcomes in infants with severe congen-
Received 27 October 2018
ital diaphragmatic hernia (CDH) after tracheal occlusion (TO) is a crucial step for advancing
Received in revised form
neonatal care. The objective of this study is to explore the heterogeneous airspace
15 January 2019
morphometry and the metabolic landscape changes in fetal lungs after TO.
Accepted 6 February 2019
Methods: Fetal lungs on days 1 and 4 after TO were examined using mass spectrometry
Available online xxx
ebased metabolomics, fluorescence lifetime imaging microscopy (FLIM), the number of airspaces, and tissue-to-airspace ratio (TAR).
Keywords:
Results: Two morphometric areas were identified in TO lungs compared with controls
Metabolic landscape
(more small airspaces at day 1 and a higher number of enlarged airspaces at day 4). Global
Heterogeneous metabolic zones
metabolomics analysis revealed a significant upregulation of glycolysis and a suppression
Tracheal occlusion
of the tricarboxylic acid cycle in day 4 TO lungs compared with day 1 TO lungs. In addition,
Pulmonary hypoplasia
there was a significant increase in polyamines involved in cell growth and proliferation.
Congenital diaphragmatic hernia
Locally, FLIM analysis on day 1 TO lungs demonstrated two types of heterogeneous
Fluorescence lifetime imaging
zonesdsimilar to control and with increased oxidative phosphorylation. FLIM on day 4 TO
microscopy
lungs demonstrated appearance of zones with enlarged airspaces and a metabolic shift toward glycolysis, accompanied by a decrease in the FLIM “lipid-surfactant” signal. Conclusions: In normal fetal lungs, we report a novel temporal pattern of varied morphometric and metabolic changes. Initially, there is formation of zones with small airspaces, followed by airspace enlargement over time. Metabolically day 1 TO lungs have zones with increased oxidative phosphorylation, whereas day 4 TO lungs have a shift toward glycolysis in the enlarged airspaces. Based on our observations, we speculate that the “best responders” to tracheal occlusion should have bigger lungs with small airspaces and normal surfactant production. ª 2019 Elsevier Inc. All rights reserved.
* Corresponding author. Division of Pediatric Surgery, Department of Surgery, University of Colorado School of Medicine, 12700 E 19th Avenue, MS 8618, Aurora, CO 80045. Tel.: þ303 724 4186; fax: þ303 724 6330. E-mail address: [email protected] (A.I. Marwan). 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.02.015
dobrinskikh et al heterogeneous lung response following to
Introduction Congenital diaphragmatic hernia (CDH) occurs in 1:2500-5000 births1,2 and results in pulmonary hypoplasia3 with different severity.4 Infants with the most severe left-sided CDH (observed-to-expected lung-to-head ratio of <25%) are currently offered an experimental interventiondtracheal occlusion (TO)dto accelerate lung growth (https://totaltrial.eu/). The technique of TO has undergone significant innovations from the initial report of “plug the lung until it grows (PLUG)” described by Hedrick et al. in 1994,5 to its current fetal endoscopic tracheal occlusion (FETO).6,7 In a few published series, reported survival rates after FETO were increased to 42%-48%;7,8 however, there have been inconsistent clinical outcomes after this intervention, the reasons of which are not fully understood. Tracheal occlusion is a classic example of how mechanosensitivity and mechanotransduction work in concert resulting in cellular proliferation and lung growth. It leverages the same mechanisms involved in pulmonary development that are dependent on the normal intrapulmonary pressure gradient generated by lung fluid production and the fixed resistance of the glottis9 accentuated by fetal breathing movements. Fetal lung development is a very complex process that follows a well-organized plan.10 Cells in the developing lungs are subjected to different forces, including tensile, compressive, hydrostatic, and fluid shear stress. The translation of local extrinsic mechanical events into fast and longstanding changes of cellular phenotype is dependent on the function of a complex cellular network of molecules and structures that are capable of sensing and responding to mechanical forces.10 Alveologenesis requires tightly regulated synchronization of multiple cell types.11,12 Thus, alveolar epithelial type 1 cells line the alveolus and form air-blood barrier, whereas alveolar epithelial type 2 cells produce and secrete surfactant, which reduces surface tension and preclude alveoli from collapsing after birth. Alveolar mesenchyme contains at least two types of fibroblasts: myofibroblasts, which produce elastin and can contract, and lipofibroblasts containing lipid droplets.13-17 Furthermore, the microvasculature of the lung contains the capillary network for gas exchange and the lymphatic network for removal of excess fluids. The interaction between mesenchymal and epithelial cells is important for cellular differentiation and function during alveolarization.18,19 Mechanical forces constitute significant stimuli for alveolarization.9,20 Surfactant production and release are stretch-dependent.21-23 Moreover, stretchdependent interactions of alveolar epithelial type 2 cells with fibroblasts can promote surfactant synthesis.22 Inhalation of amniotic fluid by prenatal breathing movements creates mechanical forces that are fundamental for the differentiation of alveolar epithelial type 1 cells.24 Also, mechanical forces can maintain mitotic spindle orientation in the airway cells and thus are important in airway morphogenesis.25 Along with other stimuli and forces, internal pressures due to fluid secretion across the pulmonary epithelium are fundamental for normal pulmonary development.9,26-28 Regulated fluid pressure has been shown to be essential for normal lung development by influencing epithelial folding and cellular proliferation.29 In addition, rapid cellular growth and proliferation require large amounts of individual building
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blocks including amino acids and nucleotides. Therefore, cells and tissues undergoing growth are expected to have metabolic changes commensurate to that dynamic process.30 It is not surprising that fetal pulmonary tissues preparing for an accelerated lung expansion after TO will shift their metabolism to glycolysis. In contrast to conventional glycolysis, actively growing cells shift the glycolytic flux toward lactate production through generation of large amounts of reduced nicotinamide adenine dinucleotide (NADH). Moreover, proliferating cells have active pentose phosphate shunt, which provides NADPH-reducing equivalents and five carbon units used to synthesize nucleotides.31-34 These processes allow for both adequate adenosine triphosphate (ATP) production and a surplus of carbon and NADH for growth.35 According to current paradigms, response of the fetal lungs to tracheal occlusion is viewed as uniform; however, we have previously suggested that the key principle in understanding changes in fetal lungs after TO is to view all concurrent changes from the perspective of biological heterogeneity.10,36 We have earlier demonstrated that after TO, the lungs will appear as a topologic mosaic characterized by a diverse metabolic landscape and parenchymal regions with variable structures in the newly formed lung tissue36; however, the natural history of changes in airspace morphometry and metabolic landscape after TO is currently unknown. The objective of this study is to examine the temporal pattern of changes in airspace morphometry and the metabolic landscape in normal fetal lungs after TO.
Material and methods Fetal rabbit tracheal occlusion model All animal procedures were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus (protocol #00434e6/2018). Time-dated pregnant New Zealand white rabbits were obtained at a gestational age of 17 to 19 d from Charles River Laboratory and housed in separate cages under standard laboratory conditions with free access to water and chow. Rabbits were allowed to acclimate for at least 5 d to compensate for the high altitude in Denver. On the day of surgery, each doe was premedicated with ketamine/xylazine (20-40 mg/kg and 5 mg/kg, respectively) and 7 mg medroxyprogesterone acetate (Depo-Provera) and were administered 300,000 units of penicillin G intramuscularly. Tracheal occlusion was performed on day 26 as described previously.36 Only two fetuses per doe were operated on, corresponding ovarianend position on opposite horns (TO and sham). When the ovarian-end fetus was smaller, a larger fetus toward the ovarian end and its corresponding position fetus on the opposite horn were selected for surgery. Fetal position was determined by gentle palpation followed by a 1-1.5 cm transverse hysterotomy on the antimesometrial border of the uterus. After delivery of the fetal head, a fetal midline neck incision was made. The trachea was dissected and separated
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from the esophagus, and a single 4-0 silk suture was passed around the trachea and gently tied in TO. A sham operation was performed on a fetus in a corresponding position on the opposite horn with exposure and dissection of the trachea, but no ligation. After surgery, fetuses were gently manipulated back into the uterine cavity. Three milliliters of warmed lactated Ringers solution was infused into the amniotic cavity to maintain amniotic fluid volume followed by closure of the hysterotomy using a running 4-0 silk suture. The uterus was placed back into the abdominal cavity and the abdomen closed in layers. All animals received bupivacaine 0.5% (12 mg/kg) and buprenorphine SR LAB (0.15 mg/kg) along the incision for postoperative analgesia in addition to 10 mL/kg of normal saline subcutaneously.
Harvest All fetuses were harvested by cesarean section on day 30. Does were subjected to the same premedication, anesthesia, and positioning. Three fetuses per doe (TO, sham, and unoperated pregnancy control) were delivered via a cesarean section. Immediate euthanasia was performed using approved methods. Successful TO was confirmed by naked eye examination, documenting larger lungs filling the chest cavity with an intact tracheal ligature and no apparent evidence of tracheal injury,
before tissue harvest. Lung tissue was immediately collected, flash-frozen in liquid nitrogen, and then stored in 80 C (for metabolomics and fluorescence lifetime imaging microscopy [FLIM]) or fixed in 4% paraformaldehyde for histological evaluation and morphometric analysis. Lung samples for morphometric analyses were not pressure-fixed as it has been previously demonstrated that morphometric indices can be compared in samples processed similarly, whether the fetal lungs are pressure- or immersion-fixed.2
Histological analysis Paraffin-embedded lungs were cut (5 mm sections) and stained with hematoxylin and eosin. Tissue sections were scanned using Aperio CS2 slide scanner (Leica Biosystems Inc, Buffalo Grove, IL) with 40 lens magnification; 20-30 random fields of view (FOVs) per animal (n ¼ 3, 3, 4; 5, 5, and 7 animals for control day 1, sham day 1, TO day 1; control day 4, sham day 4, and TO day 4, respectively, 2544 1295 pixels/image; image size was kept the same for all measurements) excluding conducting airways and major blood vessels were taken and analyzed in ImageJ (NIH, Bethesda, MD). Image acquisition and analysis were performed by different personnel (E.D. and S.I.A.-J.) with image analyses performed in a blinded fashion. Images were opened as RGB format and converted to 8-bit
Fig. 1 e TAR distribution analysis reveals at least two distinct regions in rabbit lung parenchyma after 1 d and 4 d following tracheal occlusion. (A) Hematoxylin and eosin representative images for lung tissue sections from control (top panels), sham (middle panels), and TO (bottom panels) samples at day 1 (left column) and day 4 (right column) after the surgeries are shown. In control tissues at day 1, there were a statistically significant smaller number of airspaces and tissue areas compared with day 4, which may correspond to normal lung growth. Lung tissues from sham samples 1 d after surgery showed airspaces that were similar to controls and others that were smaller (indicated by the arrows), suggesting possible induced branching as an acute response to lactate administration; however, at day 4, tissues from shams were indifferent from control samples, confirming an acute response at day 1. Lung tissues 1 d after TO demonstrated a further increase in the number of smaller airspaces (denoted by the arrows), indicating additional stimuli present in TO. With longer time (4 d) after surgery, there was an appearance of enlarged airspaces (denoted by arrowheads) in the TO samples. All TAR histograms for every condition were calculated and fitted to a gaussian fitting model. Raw data with the corresponding fits were plotted for combined histograms at day 1 (B) and day 4 (E) after surgery. Day 1 sham and TO histograms were further separated into two distributions: control-like TAR (C) and bigger TAR (smaller airspaces) (D) subsets. The day 4 sham histogram was similar to control and therefore was not separated (E-G). In contrast to day 1, the day 4 TO histogram was separated into control-like TAR (F) and smaller TAR (enlarged airspaces) (G) subsets. Student’s two-tailed t-test was used for statistical comparison. n [ 3-7 animals/group. Significantly different groups were denoted with **, P < 0.02. (Color version of figure is available online.)
dobrinskikh et al heterogeneous lung response following to
grayscale. Airspaces were thresholded using the corresponding background histogram and analyzed with Analyze Particles plugin tool. Particles greater than 500-pixel square were segmenteddcircularity range was 0-1, and holes were included. Areas of each individual particle were recovered. Consequently, tissue areas were calculated by subtracting the sum of total airspace areas from image size. The number of airspaces and tissue-to-airspace ratios (TARs) were calculated. TAR histograms were calculated and fitted to a least square Gaussian fitting model, and their means and standard deviations were extracted from the fits for statistical significance comparison purposes.
Mass spectrometryebased metabolomics analysis Global metabolomics analysis was performed on frozen lung samples from control, sham, and TO using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass spectrometer. Sample preparation, data acquisition, and analysis were performed as previously described.36 Data are graphically presented relative to the median control value at the respective time point.
Fluorescence lifetime imaging microscopy FLIM was performed to detect local metabolic changes in 15-20 different areas in a fresh lung lobe (which was kept the same for each animal) using a Zeiss 780 laser-scanning confocal/ multiphoton-excitation fluorescence microscope with a
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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). A dichroic filter (496 nm; Semrock Inc, Rochester, NY) was used to separate the fluorescence signal. The 2photon excitation was blocked by a 2-photon emission filter. For the acquisition of FLIM images, fluorescence was detected by a photon-counting photomultiplier tube detector (H7422p40; Hamamatsu Photonics, Hamamatsu, Japan). Images of the lung were obtained with the Vista Vision software by ISS in 256 256 format with a pixel dwell time of 6.3 ms/pixel and averaging over 30 frames. Calibration of the system was performed by measuring the known lifetime of the fluorophore fluorescein with a single exponential decay of 4.0 ns.37 The phasor transformation and data analysis were carried out using the Global SimFCS software (Laboratory for Fluorescence Dynamics, University of California, Irvine, CA) as described previously.38 The number of pixels covered with lifetimes for free and bound NADH, as well as “lipid-surfactant” signals, was calculated in SimFCS (Laboratory for Fluorescence Dynamics), and the values were normalized to the total number of pixels detected. Data are shown as a percentage of total in pie charts.
Statistical analysis Data were expressed as means SEM. Three-seven (n ¼ 3-7) animals/group were used. Two-sample t-test (two-tailed
Fig. 2 e Arginine metabolism is increased at day 4 after tracheal occlusion. A classical arginine metabolic pathway is accompanied by plots of metabolomics results where values are normalized to their respective day’s control group. n [ 4-7/ group. Significantly increased spermidine and spermine and decreased creatine and proline were found in TO samples 4 d after surgery. Increased spermidine and spermine levels indicate increased cell proliferation, whereas creatinine is possibly providing some required energy for cell growth. Decreased levels of fumarate and trend toward decreased levels of aspartate may indicate that arginine metabolism is shifting these intermediates toward polyamines production shunting them away from TCA cycle. Student’s t-test was used for statistical comparison between day 1 and day 4 TO groups. Significantly different metabolites are denoted with * where P < 0.05 and ** with P < 0.01.
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unpaired Student’s t-test) was performed for statistical analysis of histograms, using a 0.01 significance level and a twosided P-value. Two-sample t-test (two-tailed unpaired Student’s t-test, significance threshold P < 0.05) for metabolomics results was performed using the Prism 7.0 software (GraphPad, San Diego, CA). Statistical differences (P < 0.05) between groups for FLIM data were determined by one-way analysis of variance (Tukey correction) for multiple comparisons using the Prism 7.0 software (GraphPad).
Results Morphometric analysis reveals two unique distributions of distal airspaces in fetal rabbit lung tissues after TO To examine the natural history of parenchymal airspace morphometric changes in fetal lungs after TO, TARs were calculated at day 1 and day 4 after TO surgery and compared with their control and sham counterparts (Fig. 1). Images of hematoxylin and eosinestained lung tissue sections were used to calculate TARs for each condition, as described in the Methods Section. We found that the number of airspaces per FOV was significantly less in day 1 control samples than in day 4 control samples (83 2 at day 1 compared with 91 1 at day 4, P < 0.0005), likely corresponding to a normal lung growth pattern. At day 1, sham samples had some regions with smaller airspaces (Fig. 1A, middle left panel, denoted by arrows) than controls, whereas sham day 4 tissues were indifferent from controls (Fig. 1A, middle right panel), indicating
possible induced branching at day 1 due to an acute response to lactate administration. The number of smaller airspaces was higher in TO samples at day 1 than shams, suggesting an extra stimulation due to occlusion (Fig. 1A, bottom left panel, denoted by arrows). After 4 d of TO, enlarged airspaces were observed as shown in Figure 1A (bottom right panel, denoted by arrowheads). For each condition, a TAR histogram was calculated and fitted to a least-square Gaussian fitting model (Fig. 1B-G). Combined histograms for sham and TO samples at day 1 had a significant shift toward bigger TAR values (smaller airspaces), which was more pronounced in TO (Fig. 1B). To demonstrate the heterogeneous response to tracheal occlusion, we separated the corresponding sham and TO TAR histograms into two subdistributions: TAR values that are greater than the mean of TAR control plus one standard deviation (mean þ s) were assigned to the bigger TAR (smaller airspaces) (Fig. 1D) subdistribution, and the rest were considered as control-like TARs (Fig. 1C). These TAR subdistributions were statistically different from control and each other (Fig. 1D). On the other hand, combined histograms for TO at day 4 demonstrated a significant shift toward smaller TAR values (bigger airspaces), compared with control and sham (Fig. 1E). In a similar fashion, we separated TAR histogram values in TO samples into two subdistributions: TAR values that are smaller than the mean of TAR control minus one standard deviation (mean s) were allocated to the smaller TAR (enlarged airspaces) (Fig. 1G) subdistribution, and the remaining values were considered as similar to control (Fig. 1F). TO small TAR subdistribution was statistically different from control and sham TAR distributions (Fig. 1G).
Fig. 3 e Tricarboxylic acid (TCA) cycle is suppressed at day 4 after TO. In all graphs, metabolic data are shown relative to control. n [ 4-7/group. Metabolic analysis demonstrated a decline in the TCA cycle with a significant decrease in citrate and fumarate in day 4 compared with day 1 TO lungs. This suggests a high demand for energy and metabolite production and a shift to faster glycolysis. Student’s two-tailed t-test was used for statistical comparison between day 1 and day 4 TO groups. Significantly different metabolites are denoted with ** where P < 0.01.
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Fig. 4 e Glycolysis is upregulated in the fetal lungs at day 4 after TO. In all graphs, metabolic data are shown as relative to control. n [ 4-7/group. Glucose and hexose phosphate were significantly decreased in day 4 compared with day 1 TO lungs. This was accompanied by a significant shift toward lactate production at day 4 after TO compared with day 1. Student’s twotailed t-test was used for statistical comparison between day 1 and day 4 TO groups. Significantly different metabolites are denoted with * where P < 0.05.
Day 4 sham TAR distribution was not statistically different from that of day 4 control and thereby was not separated into the two subdistributions mentioned previously.
The global metabolic landscape after TO Metabolism impacts all cellular functions, including cell proliferation and differentiation required for lung development. Untargeted high-throughput metabolomics was performed using ultra high-pressure liquid chromatography coupled online to mass spectrometry (UHPLC-MS) and demonstrated a shift from mitochondrial metabolism to glycolysis 4 d after TO in comparison with sham and control, indicating high demands for energy in occluded lungs.
Upregulated arginine metabolism at day 4 after TO Arginine cycle metabolites play important roles in multiple pathways (a schematic presentation of a classical arginine metabolic pathway is depicted in Fig. 2); for example, creatine is necessary for ATP production,39 and ornithine is involved in cell proliferation, extracellular matrix (ECM) remodeling, and nitrogen excretion via the urea cycle. For cell proliferation processes, ornithine is converted to putrescine followed by spermidine and spermine.40 Conversion to proline is essential
for ECM proteins that are rich in proline and glycine production, such as collagens and elastin.41 We have found significantly increased levels of spermidine and spermine (Fig. 2) and a trend toward a decrease in proline and creatine in day 1 TO lungs compared with day 4 TO lungs. In addition, we have found statistically indifferent levels of aspartate and decreased fumarate in day 1 TO lungs compared with day 4 TO lungs (Fig. 2), which might indicate that arginine metabolism is shifting these intermediates toward polyamine production, channeling them away from the tricarboxylic acid (TCA) cycle.
TCA cycle suppression and glycolysis upregulation at day 4 after TO Both the TCA cycle (Fig. 3) and glycolysis (Fig. 4) play crucial roles in ATP production, but their speeds and efficiencies are different42 and metabolic flexibility is very important for adaptation to different microenvironmental conditions.43 Differentiated cells mostly use oxidative phosphorylation (OXPHOS) to efficiently generate necessary amounts of ATP,30,44 whereas rapidly proliferating cells and cells devoid of adequate oxygen shift to increased nutrient uptake and glycolysis.30 Metabolomics analysis revealed a decline in the TCA cycle ascertained by a significant decrease in citrate and fumarate in day 4 TO lungs compared with day 1 TO lungs (Fig. 3). In addition, glucose
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Fig. 5 e Mitochondrial transport and fatty acid metabolism. In all graphs, metabolic data are shown as relative to control. n [ 4-7/group. Caprylate (C 8:0) and pelargonate (C 9:0) were decreased at day 4 after TO. Most metabolites involved in the carnitine shuttle were increased at day 4 after TO compared with day 1. Student’s two-tailed t-test was used for statistical comparison between day 1 and day 4 TO groups. Significantly different metabolites are denoted with * where P < 0.05 and ** with P < 0.01.
and hexose phosphate levels were significantly decreased in day 4 TO lungs compared with day 1 TO lungs (Fig. 4). This was accompanied by a significant shift toward lactate production in day 4 TO lungs compared with day 1 TO lungs (Fig. 4), suggesting the use of ongoing glycolysis to meet the increased needs for energy production in occluded lungs.
Mitochondrial transport and fatty acids beta-oxidation are altered in TO lungs Fatty acid beta-oxidation occurs primarily in the mitochondria and is important for ATP and NADH generation.45 It also generates the TCA cycle metabolite acetyl coenzyme A (acetylCoA). To enter mitochondria, fatty acids attached to coenzyme A (acyl-CoA) are shuttled into the mitochondria as acylcarnitine intermediates46,47 (Fig. 5). Following translocase activity, acylcarnitines are converted back to their acyl-CoA forms, allowing beta-oxidation to occur. We found that the level of caprylate (C 8:0) was significantly decreased and pelargonate (C 9:0) had a trend to decrease at day 4 after TO (Fig. 5). Four days after TO, there was an observed increase in most metabolites involved in the carnitine shuttle compared with day 1 (Fig. 5).
The local metabolic landscape after tracheal occlusion Metabolic changes at day 1 after TO are consistent with an increase in oxidative phosphorylation FLIM was used to examine local metabolic changes in fetal lungs at day 1 and day 4 after TO. Although intensity images of
multiple random FOVs at day 1 were similar in all examined groups of animals (Fig. 6A, top panel), corresponding FLIM maps showed heterogeneous lifetime allocations in the TO group, compared with sham and control. Lifetime distribution in day 1 TO lungs revealed two distinct regionsdregions similar to control and regions with a visually increased lipid-surfactant signal (Fig. 6A, bottom panel). Quantification of percentages covered by specific lifetime signals and free/bound NADH ratios in control and sham animals demonstrated a nonstatistically significant decrease in the NADH lifetime percentages (Fig. 6B, 9 1% for free NADH and 58 2% for bound NADH in shams versus 11 2% for free NADH and 63 2% for bound NADH in controls) and the free/bound NADH ratio in shams compared with controls (Fig. 6C, 0.16 0.02 compared with 0.21 0.04, respectively). On the other hand, both free and bound NADH lifetime percentages (Fig. 6B, 7 1% for free NADH and 49 2% for bound NADH in TO versus 11 2% for free NADH and 63 2% for bound NADH in controls, P < 0.0001) and free/bound NADH ratio were significantly decreased in combined analysis of all images from day 1 TO lungs compared with controls (Fig. 6C, 0.11 0.01 versus 0.21 0.04, respectively, P ¼ 0.0017). In addition to decreased free/bound NADH ratio, indicating a shift toward OXPHOS,48 there was an increase in the percentage of pixels with lipidsurfactant signals (Fig. 6B, cyan and magenta sectors), suggesting a possible surfactant release, perhaps as a response to the initial mechanical stretch stimulus after TO surgery; however, after the separation of regions with dissimilar FLIM maps from the occluded fetal lungs, we demonstrated that a decrease in
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Fig. 6 e Local metabolic landscape demonstrates the presence of at least two different regions after tracheal occlusion. (A) Representative intensity images (top row) with corresponding FLIM maps (bottom row) of control, sham, and TO lungs at day 1 after surgery are shown. Fetal lungs in all conditions demonstrated similar structures; however, the local metabolic landscape was heterogenous in lungs after TO. (B) Graphical representation of percentage covered by specific lifetime signals in controls and sham animals exhibited a nonstatistically significant decrease in the free/bound NADH ratio in shams compared with controls. Combined regions of TO lungs showed a significant decrease in the free/bound NADH ratio (C) and an increased lipidsurfactant signal, indicating a surfactant release and shift toward OXPHOS, probably as a response to initial mechanical stretch after TO surgery. Separation of zones based on their FLIM maps revealed presence of zones with similar to control signatures and a more pronounced significant decrease in the free/bound NADH ratio (D) with an increased lipid-surfactant signal. (E) Representative intensity images (top row) with corresponding FLIM maps (bottom row) of control, sham, and TO lungs at day 4 after surgery are shown. Control and sham lungs displayed similar structures with a homogeneous metabolic landscape. Conversely, TO demonstrated two distinct types of zones; one type of zones was structurally similar to both controls and shams and had a similar free/bound NADH ratio (H). The second type of zones in TO demonstrated evidence of airspaces enlargement and is characterized by a significant shift toward glycolysis and an decreased lipid-surfactant signal. (F) Graphical representation of percentage covered by specific lifetime signals in controls, shams, and combined regions of TO animals demonstrated a nonstatistically significant increase in the free/bound NADH ratio (G) in shams compared with controls. One-way analysis of variance (Tukey correction) was used for statistical comparison between the groups. n [ 4-7 animals/group. Statistical differences were determined as significant for P < 0.05. (Color version of figure is available online.)
free/bound NADH ratio (Fig. 6B, TO separated; Fig. 6D red shaded bar, TO OXPHOS [0.07 0.01]) was observed in the areas with a increased lipid-surfactant signal, whereas the remaining zones were similar to shams and controls (Fig. 6B, TO separated; Fig. 6D purple shaded bar, TO control [0.22 0.03]). In contrast to histological findings, local metabolic changes in day 1 sham lungs were not different from controls (Fig. 6A-C), indirectly confirming a possible acute transient effect secondary to intra-amniotic lactate instillation.
Enlarged airspaces 4 d after TO are associated with a shift toward glycolysis Similar to the histological changes, at day 4 after occlusion, we observed heterogeneous intensity images: images with controllike and images with enlarged parenchymal structures,
whereas sham and control intensity images were indifferent from each other (Fig. 6E, top panel). Corresponding FLIM maps revealed a unique increase in free NADH lifetime in regions with enlarged structures in TO lungs (Fig. 6E, bottom panel), indicating a shift toward glycolysis48 in these areas. Quantification of percentages covered by particular lifetime signals in control and sham animals demonstrated similar percentages for free and bound NADH lifetimes (Fig. 6F, 28 2% for free NADH and 62 1% for bound NADH in shams versus 21 1% for free NADH and 73 2% for bound NADH in controls) and a nonstatistically significant increase in the free/bound NADH ratio in shams compared with controls (Fig. 6G, 0.53 0.05 versus 0.35 0.08, respectively). Analysis of all images taken in animals after TO demonstrated a significant increase of free (and decrease of bound) NADH lifetime percentage(s) (Fig. 6F, 39
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Fig. 7 e Model of lung development after tracheal occlusion (TO). The five well-known stages for normal lung development, their major characterizing events, and corresponding rabbit gestational ages adapted from Surate Solaligue et al.52 are depicted. TO is introduced at E26, which leads to accumulation of fluid containing different stimuli, including growth factors and metabolites. Our morphometric findings (highlighted with blue font) showeddin addition to control-likedan appearance of smaller airspaces at day 1 after TO. This corresponded to greater TAR values, suggesting an increased branching, whereas 4 d after TO, there is an appearance of enlarged (highlighted with red font and arrow) airspaces resulted in reduced TAR values, presumably induced by increased fluid accumulation in these regions and control-like TAR values (highlighted with green font and arrows). The natural history of the local metabolic FLIM signals (highlighted with brown font) suggests that there is an increase in OXPHOS and “lipid-surfactant” signals in some areas at day 1 after the occlusion, relative to control; however, the “lipid-surfactant” FLIM signal is reduceddand accompanied by a significant metabolic shift toward glycolysisdin the enlarged airspaces at day 4 after TO, whereas control-like airspaces, at day 4, maintain both a normal “lipid-surfactant” FLIM signal and a balanced metabolism. Based on our observations and assumptions, we would anticipate that the “best responders” to tracheal occlusion should have bigger lungs with more control-like airspaces maintaining normal surfactant production. (Color version of figure is available online.)
2% for free NADH and 58 2% for bound NADH in TO versus 21 1% for free NADH and 73 2% for bound NADH in controls, P < 0.0001) and a significant free/bound NADH ratio increase in day 4 TO lungs compared with controls (Fig. 6G, 0.92 0.11 versus 0.35 0.08, respectively, P ¼ 0.0006). Moreover, there was a substantial decrease in the percentage of pixels with lipidsurfactant signals in all group of animals, compared with day 1 counterparts (Fig. 6F versus Fig. 6B), perhaps due to observed overall increased TAR values (i.e., smaller airspaces) for day 4 lungs compared with day 1 lungs (Fig. 1E versus Fig. 1B). The TO group had a further decrease in the lipid-surfactant signal, which agrees with the decrease in surfactant production reported previously.49,50 After separation of regions with dissimilar structures, from the occluded fetal lungs, we observed an increased free/bound NADH ratio and an almost absent lipid-surfactant signal in the areas with enlarged airspaces (Fig. 6F, TO separated; Fig. 6H red bar, TO glycolysis [1.45 0.19]), indicating a shift toward glycolysis possibly due to a higheenergy demand environment.
Discussion In this article, we describe the temporal pattern of the heterogeneous airspace morphometry and the metabolic
landscape changes in normal fetal lungs undergoing TO. Previously, we have proposed and demonstrated the heterogeneity concept of TO lung growth by virtue of the local metabolic landscape using FLIM on lung lobes at day 4.36 Here, we used morphometric measures to confirm our findings on the histological level, explored the natural history of the morphometric heterogeneity after TO, and described the global and local metabolic changes. Overall, the number of airspaces and tissue areas were less in day 1 control samples than day 4 samples, indicating normal lung growth. Our observation of an increase in smaller airspaces in day 1 sham samples may be attributed to an induced branching at day 1 due to an acute response to lactate administration upon hysterotomy closure. With regard to global changes in metabolism after TO, it is not surprising that by day 4, there was a shift away from TCA and toward glycolysis. This finding is in agreement with a “Warburg-like” metabolism being adopted by actively proliferating tissues to support growth30; however, a possible explanation for the early (day 1) local metabolic FLIM findings may be related to the stretch of type 2 cells in response to mechanical stimulation after occlusion. This correlates with the OXPHOS-driven metabolic landscape at day 1. Consistent with the anticipated stretch of type 2 cells, it is expected that there will be an increase in the surfactant signal as seen in our
dobrinskikh et al heterogeneous lung response following to
day 1 FLIM data; however, as occlusion is maintained for 4 d, cells will start to divide, and therefore a shift in the metabolic landscape toward glycolysis is needed. At day 4, the FLIM lipid-surfactant signal decreased, suggesting a decrease in surfactant production, which was previously shown in proliferating type 2 cells.51 Another explanation for the reduction in surfactant production, suggested by De Paepe et al., may be attributed to an accelerated terminal differentiation of type 2 to type 1 cells.49 One interesting observation with regard to the FLIM lipidsurfactant signal is the lower baseline FLIM signal in the day 4 controls compared with day 1 controls. This reduction can be explained by the expected lung growth from day 26 to day 30 (which will result in more tissue, i.e., more NADH FLIM signal compared with “lipid-surfactant” counterpart) and, therefore, the relative percentage decrease of the surfactant signal. Although we showed that changes in FLIM signals were observed in enlarged airspaces, it is currently unknown whether these FLIM lipid-surfactant signals are associated with changes in ECM proteins production to support alveolar wall. Furthermore, it will be important to examine the correlation between these changes and the appearance of heterogeneous histological and metabolic zones. Although these observations are fundamental in understanding the metabolic response of normal lungs to occlusion, it will be important to examine the metabolic landscape in fetuses who have undergone a surgical creation of a CDH followed by TO. These experiments are currently underway in our laboratory.
Conclusions In conclusion, in this article, we confirm our concept of formation of heterogeneous zones in occluded fetal lungs and describe the natural history of the morphometric and metabolic changes after TO (Fig. 7). Initially there is formation of zones with small airspaces, followed by airspace enlargement over time. Metabolically, day 1 TO lungs have zones with increased OXPHOS, whereas day 4 TO lungs have a shift toward glycolysis in the enlarged airspaces. Thus, we highlight the relationship between metabolic and morphometric changes to prime the pulmonary machinery for mechano-dependent lung growth.
Acknowledgment The authors thank the University of Colorado School of Medicine, the University of Colorado Veterinary services for their assistance with animal surgeries and postsurgical care, and the Morphology Core for their assistance with sample preparation and processing. Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core supported in part by NIH/ NCATS Colorado CTSI Grant Number UL1 TR001082. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. Authors’ contribution: E.D. conceptualized the study; performed the histological staining and imaging and FLIM imaging
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and analyses; analyzed and interpreted the data; and contributed to manuscript writing, editing, and critical review. S.I.A.-J. performed histological analysis, histograms fitting, and statistical comparisons and contributed to manuscript writing, editing, and critical review. U.S. and A.I.M. conceptualized the study, performed the animal surgeries, analyzed and interpreted the data, and contributed to manuscript writing, editing, and critical review. J.A.R. and C.Z. performed metabolomics and contributed to manuscript editing and critical review.
Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.
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