Acute-on-chronic: using magnetic resonance imaging to disentangle the haemodynamic responses to acute and chronic fetal hypoxaemia.

Introduction: The fetal haemodynamic response to acute episodes of hypoxaemia are well characterised. However, how these responses change when the hypoxaemia becomes more chronic in nature such as that associated with fetal growth restriction (FGR), is less well understood. Herein, we utilised a combination of clinically relevant MRI techniques to comprehensively characterize and differentiate the haemodynamic responses occurring during acute and chronic periods of fetal hypoxaemia. Methods: Prior to conception, carunclectomy surgery was performed on non-pregnant ewes to induce FGR. At 108-110 days (d) gestational age (GA), pregnant ewes bearing control (n = 12) and FGR (n = 9) fetuses underwent fetal catheterisation surgery. At 117-119 days GA, ewes underwent MRI sessions where phase-contrast (PC) and T2 oximetry were used to measure blood flow and oxygenation, respectively, throughout the fetal circulation during a normoxia and then an acute hypoxia state. Results: Fetal oxygen delivery (DO2) was lower in FGR fetuses than controls during the normoxia state but cerebral DO2 remained similar between fetal groups. Acute hypoxia reduced both overall fetal and cerebral DO2. FGR increased ductus venosus (DV) and foramen ovale (FO) blood flow during both the normoxia and acute hypoxia states. Pulmonary blood flow (PBF) was lower in FGR fetuses during the normoxia state but similar to controls during the acute hypoxia state when PBF in controls was decreased. Conclusion: Despite a prevailing level of chronic hypoxaemia, the FGR fetus upregulates the preferential streaming of oxygen-rich blood via the DV-FO pathway to maintain cerebral DO2. However, this upregulation is unable to maintain cerebral DO2 during further exposure to an acute episode of hypoxaemia. The haemodynamic alterations required at the level of the liver and lung to allow the DV-FO pathway to maintain cerebral DO2, may have lasting consequences on hepatic function and pulmonary vascular regulation after birth.

Post-Myocardial Infarction Remodeling and Hyperkinetic Remote Myocardium in Sheep Measured by Cardiac MRI Feature Tracking.

BACKGROUND: Cardiac MRI feature tracking (FT) allows objective assessment of segmental left ventricular (LV) function following a myocardial infarction (MI), but its utilization in sheep, where interventions can be tested, is lacking. PURPOSE: To apply and validate FT in a sheep model of MI and describe post-MI LV remodeling. STUDY TYPE: Animal model, longitudinal. ANIMAL MODEL: Eighteen lambs (6 months, male, n = 14; female, n = 4; 25.2 ± 4.5 kg). FIELD STRENGTH/SEQUENCE: Two-dimensional balanced steady-state free precession (bSSFP) and 3D inversion recovery fast low angle shot (IR-FLASH) sequences at 3 T. ASSESSMENT: Seven lambs underwent test-retest imaging to assess FT interstudy reproducibility. MI was induced in the remaining 11 by coronary ligation with MRI being undertaken before and 15 days post-MI. Injury size was measured by late gadolinium enhancement (LGE) and LV volumes, LV mass, ejection fraction (LVEF), and wall thickness (LVWT) were measured, with FT measures of global and segmental radial, circumferential, and longitudinal strain. STATISTICAL TESTS: Sampling variability, inter-study, intra and interobserver reproducibility were assessed using Pearson's correlation, Bland-Altman analyses, and intra-class correlation coefficients (ICC). Diagnostic performance of segmental strain to predict LGE was assessed using receiver operating characteristic curve analysis. Significant differences were considered P < 0.05. RESULTS: Inter-study reproducibility of FT was overall good to excellent, with global strain being more reproducible than segmental strain (ICC = 0.89-0.98 vs. 0.77-0.96). MI (4.0 ± 3.7% LV mass) led to LV remodeling, as evident by significantly increased LV volumes and LV mass, and significantly decreased LVWT in injured regions, while LVEF was preserved (54.9 ± 6.9% vs. 55.6 ± 5.7%; P = 0.778). Segmental circumferential strain (CS) correlated most strongly with LGE. Basal and mid- CS increased significantly, while apical CS significantly decreased post-MI. DATA CONCLUSION: FT is reproducible and compensation by hyperkinetic remote myocardium may manifest as overall preserved global LV function. EVIDENCE LEVEL: N/A TECHNICAL EFFICACY: Stage 2.

Echocardiographic assessment of cardiovascular physiology of preterm miniature piglets supported with a pumped artificial placenta system.

OBJECTIVES: We evaluated fetal cardiovascular physiology and mode of cardiac failure in premature miniature piglets on a pumped artificial placenta (AP) circuit. METHODS: Fetal pigs were cannulated via the umbilical vessels and transitioned to an AP circuit composed of a centrifugal pump and neonatal oxygenator and maintained in a fluid-filled biobag. Echocardiographic studies were conducted to measure ventricular function, umbilical blood flow, and fluid status. In utero scans were used as control data. RESULTS: AP fetuses (n = 13; 102±4d gestational age [term 115d]; 616 ± 139 g [g]; survival 46.4 ± 46.8 h) were tachycardic and hypertensive with initially supraphysiologic circuit flows. Increased myocardial wall thickness was observed. Signs of fetal hydrops were present in all piglets. Global longitudinal strain (GLS) measurements increased in the left ventricle (LV) after transition to the circuit. Right ventricle (RV) and LV strain rate decreased early during AP support compared with in utero measurements but recovered toward the end of the experiment. Fetuses supported for >24 h had similar RV GLS to in utero controls and significantly higher GLS compared to piglets surviving only up to 24 h. CONCLUSIONS: Fetuses on a pump-supported AP circuit experienced an increase in afterload, and redistribution of blood flow between the AP and systemic circulations, associated with elevated end-diastolic filling pressures. This resulted in heart failure and hydrops. These preterm fetuses were unable to tolerate the hemodynamic changes associated with connection to the current AP circuit. To better mimic the physiology of the native placenta and preserve normal fetal cardiovascular physiology, further optimization of the circuit will be required.

Maternal tadalafil treatment does not increase uterine artery blood flow or oxygen delivery in the pregnant ewe.

Increasing placental perfusion (PP) could improve outcomes of growth-restricted fetuses. One way of increasing PP may be by using phosphodiesterase (PDE)-5 inhibitors, which induce vasodilatation of vascular beds. We used a combination of clinically relevant magnetic resonance imaging (MRI) techniques to characterize the impact that tadalafil infusion has on maternal, placental and fetal circulations. At 116-117 days' gestational age (dGA; term, 150 days), pregnant ewes (n = 6) underwent fetal catheterization surgery. At 120-123 dGA ewes were anaesthetized and MRI scans were performed during three acquisition windows: a basal state and then ∼15-75 min (TAD 1) and ∼75-135 min (TAD 2) post maternal administration (24 mg; intravenous bolus) of tadalafil. Phase contrast MRI and T2 oximetry were used to measure blood flow and oxygen delivery. Placental diffusion and PP were assessed using the Diffusion-Relaxation Combined Imaging for Detailed Placental Evaluation-'DECIDE' technique. Uterine artery (UtA) blood flow when normalized to maternal left ventricular cardiac output (LVCO) was reduced in both TAD periods. DECIDE imaging found no impact of tadalafil on placental diffusivity or fetoplacental blood volume fraction. Maternal-placental blood volume fraction was increased in the TAD 2 period. Fetal D O 2 ${D_{{{\mathrm{O}}_2}}}$ and V ̇ O 2 ${\dot V_{{{\mathrm{O}}_2}}}$ were not affected by maternal tadalafil administration. Maternal tadalafil administration did not increase UtA blood flow and thus may not be an effective vasodilator at the level of the UtAs. The increased maternal-placental blood volume fraction may indicate local vasodilatation of the maternal intervillous space, which may have compensated for the reduced proportion of UtA D O 2 ${D_{{{\mathrm{O}}_2}}}$ .

Pulmonary Vascular Regulation in the Fetal and Transitional Lung.

Fetal lungs have fewer and smaller arteries with higher pulmonary vascular resistance (PVR) than a newborn. As gestation advances, the pulmonary circulation becomes more sensitive to changes in pulmonary arterial oxygen tension, which prepares them for the dramatic drop in PVR and increase in pulmonary blood flow (PBF) that occur when the baby takes its first few breaths of air, thus driving the transition from fetal to postnatal circulation. Dynamic and intricate regulatory mechanisms control PBF throughout development and are essential in supporting gas exchange after birth. Understanding these concepts is crucial given the role the pulmonary vasculature plays in the development of complications with transition, such as in the setting of persistent pulmonary hypertension of the newborn and congenital heart disease. An improved understanding of pulmonary vascular regulation may reveal opportunities for better clinical management.

Reduced in utero substrate supply decreases mitochondrial abundance and alters the expression of metabolic signalling molecules in the fetal sheep heart.

Babies born with fetal growth restriction (FGR) are at higher risk of developing cardiometabolic diseases across the life course. The reduction in substrate supply to the developing fetus that causes FGR not only alters cardiac growth and structure but may have deleterious effects on metabolism and function. Using a sheep model of placental restriction to induce FGR, we investigated key cardiac metabolic and functional markers that may be altered in FGR. We also employed phase-contrast magnetic resonance imaging MRI to assess left ventricular cardiac output (LVCO) as a measure of cardiac function. We hypothesized that signalling molecules involved in cardiac fatty acid utilisation and contractility would be impaired by FGR and that this would have a negative impact on LVCO in the late gestation fetus. Key glucose (GLUT4 protein) and fatty acid (FATP, CD36 gene expression) substrate transporters were significantly reduced in the hearts of FGR fetuses. We also found reduced mitochondrial numbers as well as abundance of electron transport chain complexes (complexes II and IV). These data suggest that FGR diminishes metabolic and mitochondrial capacity in the fetal heart; however, alterations were not correlated with fetal LVCO. Overall, these data show that FGR alters fetal cardiac metabolism in late gestation. If sustained ex utero, this altered metabolic profile may contribute to poor cardiac outcomes in FGR-born individuals after birth. KEY POINTS: Around the time of birth, substrate utilisation in the fetal heart switches from carbohydrates to fatty acids. However, the effect of fetal growth restriction (FGR) on this switch, and thus the ability of the fetal heart to effectively metabolise fatty acids, is not fully understood. Using a sheep model of early onset FGR, we observed significant downregulation in mRNA expression of fatty acid receptors CD36 and FABP in the fetal heart. FGR fetuses also had significantly lower cardiac mitochondrial abundance than controls. There was a reduction in abundance of complexes II and IV within the electron transport chain of the FGR fetal heart, suggesting altered ATP production. This indicates reduced fatty acid metabolism and mitochondrial function in the heart of the FGR fetus, which may have detrimental long-term implications and contribute to increased risk of cardiovascular disease later in life.

Feasibility of MRI assessment of maternal-fetal oxygen transport and consumption relative to maternal position in healthy late gestational pregnancies.

Late gestational supine positioning reduces maternal cardiac output due to inferior vena caval (IVC) compression, despite increased collateral venous return. However, little is known about the impact of maternal position on oxygen (O2 ) delivery and consumption of the gravid uterus, fetus, placenta and lower limbs. We studied the effects of maternal positioning on these parameters in 20 healthy pregnant subjects at 36 ± 2 weeks using magnetic resonance imaging (MRI); a follow-up MRI was performed 6-months postpartum (n = 16/20). MRI techniques included phase-contrast and T1/T2 relaxometry for blood flow and oximetry imaging, respectively. O2 transport was measured in the following vessels (bilateral where appropriate): maternal abdominal descending aorta (DAoabdo ), IVC, ovarian, paraspinal veins (PSV), uterine artery (UtA) and external iliacs, and umbilical. Maternal cardiac output was measured by summing DAothoracic and superior vena cava flows. Supine mothers (n = 6) had lower cardiac output and O2 delivery in the DAoabdo , UtA and external iliac arteries, and higher PSV flow than those in either the left (n = 8) or right (n = 6) lateral positions during MRI. However, O2 consumption in the gravid uterus, fetus, placenta and lower limbs was unaffected by maternal positioning. The ratio of IVC/PSV flow decreased in supine mothers while ovarian venous flow and O2 saturation were unaltered, suggesting a major route of pelvic venous return unaffected by maternal position. Placental-fetal O2 transport and consumption were similar between left and right lateral maternal positions. In comparison to non-pregnant findings, DAoabdo and UtA O2 delivery and pelvic O2 consumption increased, while lower-limb consumption remained constant , despite reduced external iliac artery O2 delivery in late gestation. KEY POINTS: Though sleeping supine during the third trimester is associated with an increased risk of antepartum stillbirth, the underlying biological mechanisms are not fully understood. Maternal cardiac output and uteroplacental flow are reduced in supine mothers due to inferior vena caval compression from the weight of the gravid uterus. This MRI study provides a comprehensive circulatory assessment, demonstrating reduced maternal cardiac output and O2 delivery (uteroplacental, lower body) in supine compared to lateral positioning; however, O2 consumption (gravid uterus, fetus, placenta, lower limbs) was preserved. Unlike other mammalian species, the ovarian veins conduct substantial venous return from the human pregnant uterus that is unaffected by maternal positioning. Lumbar paraspinal venous flow increased in supine mothers. These observations may have important considerations during major pelvic surgery in pregnancy (i.e. placenta percreta). Future studies should address the importance of maternal positioning as a potential tool to deliver improved perinatal outcomes in pregnancies with compromised uteroplacental O2 delivery.

Cardiac growth and metabolism of the fetal sheep are not vulnerable to a 10 day increase in fetal glucose and insulin concentrations during late gestation.

Aims: To evaluate the effects of fetal glucose infusion in late gestation on the mRNA expression and protein abundance of molecules involved in the regulation of cardiac growth and metabolism. Main methods: Either saline or glucose was infused into fetal sheep from 130 to 140 days (d) gestation (term, 150 d). At 140 d gestation, left ventricle tissue samples were collected. Quantitative real-time RT-PCR and Western blot were used to determine the mRNA expression and protein abundance of key signalling molecules within the left ventricle of the fetal heart. Key findings: Although intra-fetal glucose infusion increased fetal plasma glucose and insulin concentrations, there was no change in the expression of molecules within the signalling pathways that regulate proliferation, hypertrophy, apoptosis or fibrosis in the fetal heart. Cardiac Solute carrier family 2 member 1 (SLC2A1) mRNA expression was decreased by glucose infusion. Glucose infusion increased cardiac mRNA expression of both Peroxisome proliferator activated receptor alpha (PPARA) and peroxisome proliferator activated receptor gamma (PPARG). However, there was no change in the mRNA expression of PPAR cofactors or molecules with PPAR response elements. Furthermore, glucose infusion did not impact the protein abundance of the 5 oxidative phosphorylation complexes of the electron transport chain. Significance: Despite a 10-day doubling of fetal plasma glucose and insulin concentrations, the present study suggests that within the fetal left ventricle, the mRNA and protein expression of the signalling molecules involved in cardiac growth, development and metabolism are relatively unaffected.

Acute resveratrol exposure does not impact hemodynamics of the fetal sheep.

Babies born growth restricted are at an increased risk of both poor short-and long-term outcomes. Current interventions to improve fetal growth are ineffective and do not lower the lifetime risk of poor health status. Maternal resveratrol (RSV) treatment increases uterine artery blood flow, fetal oxygenation, and fetal weight. However, studies suggest that diets high in polyphenols such as RSV may impair fetal hemodynamics. We aimed to characterize the effect of RSV on fetal hemodynamics to further assess its safety as an intervention strategy. Pregnant ewes underwent magnetic resonance imaging (MRI) scans to measure blood flow and oxygenation within the fetal circulation using phase contrast-MRI and T2 oximetry. Blood flow and oxygenation measures were performed in a basal state and then repeated while the fetus was exposed to RSV. Fetal blood pressure and heart rate were not different between states. RSV did not impact fetal oxygen delivery (DO2 ) or consumption (VO2 ). Blood flow and oxygen delivery throughout the major vessels of the fetal circulation were not different between basal and RSV states. As such, acute exposure of the fetus to RSV does not directly impact fetal hemodynamics. This strengthens the rationale for the use of RSV as an intervention strategy against fetal growth restriction.

Advanced imaging of fetal cardiac function.

Over recent decades, a variety of advanced imaging techniques for assessing cardiovascular physiology and cardiac function in adults and children have been applied in the fetus. In many cases, technical development has been required to allow feasibility in the fetus, while an appreciation of the unique physiology of the fetal circulation is required for proper interpretation of the findings. This review will focus on recent advances in fetal echocardiography and cardiovascular magnetic resonance (CMR), providing examples of their application in research and clinical settings. We will also consider future directions for these technologies, including their ongoing technical development and potential clinical value.

Automated Proteomics Workflows for High-Throughput Library Generation and Biomarker Detection Using Data-Independent Acquisition.

Sequential window acquisition of all theoretical mass spectra-mass spectrometry underpinned by advanced bioinformatics offers a framework for comprehensive analysis of proteomes and the discovery of robust biomarkers. However, the lack of a generic sample preparation platform to tackle the heterogeneity of material collected from different sources may be a limiting factor to the broad application of this technique. We have developed universal and fully automated workflows using a robotic sample preparation platform, which enabled in-depth and reproducible proteome coverage and characterization of bovine and ovine specimens representing healthy animals and a model of myocardial infarction. High correlation (R2 = 0.85) between sheep proteomics and transcriptomics datasets validated the developments. The findings suggest that automated workflows can be employed for various clinical applications across different animal species and animal models of health and disease.

A change of heart: understanding the mechanisms regulating cardiac proliferation and metabolism before and after birth.

Mammalian cardiomyocytes undergo major maturational changes in preparation for birth and postnatal life. Immature cardiomyocytes contribute to cardiac growth via proliferation and thus the heart has the capacity to regenerate. To prepare for postnatal life, structural and metabolic changes associated with increased cardiac output and function must occur. This includes exit from the cell cycle, hypertrophic growth, mitochondrial maturation and sarcomeric protein isoform switching. However, these changes come at a price: the loss of cardiac regenerative capacity such that damage to the heart in postnatal life is permanent. This is a significant barrier to the development of new treatments for cardiac repair and contributes to heart failure. The transitional period of cardiomyocyte growth is a complex and multifaceted event. In this review, we focus on studies that have investigated this critical transition period as well as novel factors that may regulate and drive this process. We also discuss the potential use of new biomarkers for the detection of myocardial infarction and, in the broader sense, cardiovascular disease.

MRI Characterization of Blood Flow and Oxygen Delivery in the Fetal Sheep whilst Exposed to Sildenafil Citrate.

INTRODUCTION: Newborns exposed to sildenafil citrate (SC) in utero have increased rates of persistent pulmonary hypertension. The mechanism behind this has not yet been fully elucidated. We aimed to utilize a combination of clinically relevant MRI techniques to comprehensively characterize the haemodynamics of the fetal sheep whilst under the influence of SC. We hypothesized that these MRI techniques would detect SC-induced increases in pulmonary blood flow and oxygen delivery prior to birth. METHODS: At 116-117 days gestational age (term, 150 days), pregnant Merino ewes (n = 9) underwent fetal catheterization surgery. MRI scans were performed during a basal state and then repeated during a constant umbilical vein infusion of SC to measure blood flow and oxygenation within the major vessels of the fetal circulation using phase-contrast-MRI and T2 oximetry. RESULTS: Right and left ventricular cardiac outputs were not different between states. Pulmonary blood flow increased during the SC state resulting in elevated pulmonary oxygen delivery. Right to left heart shunting through the foramen ovale was reduced without reducing cerebral oxygen delivery. CONCLUSION: SC induces alterations to pulmonary haemodynamics in utero; a characteristic that if maintained may underlie or act as a precursor towards the elevated rates of poor pulmonary outcomes after birth. These MRI techniques are the first to comprehensively characterize sildenafil's direct impact on the pulmonary vasculature and its indirect detriment to the flow of oxygen-rich blood through the foramen ovale.

Placental MRI Predicts Fetal Oxygenation and Growth Rates in Sheep and Human Pregnancy.

Magnetic resonance imaging (MRI) assessment of fetal blood oxygen saturation (SO2 ) can transform the clinical management of high-risk pregnancies affected by fetal growth restriction (FGR). Here, a novel MRI method assesses the feasibility of identifying normally grown and FGR fetuses in sheep and is then applied to humans. MRI scans are performed in pregnant ewes at 110 and 140 days (term = 150d) gestation and in pregnant women at 28+3  ± 2+5 weeks to measure feto-placental SO2 . Birth weight is collected and, in sheep, fetal blood SO2 is measured with a blood gas analyzer (BGA). Fetal arterial SO2 measured by BGA predicts fetal birth weight in sheep and distinguishes between fetuses that are normally grown, small for gestational age, and FGR. MRI feto-placental SO2 in late gestation is related to fetal blood SO2 measured by BGA and body weight. In sheep, MRI feto-placental SO2 in mid-gestation is related to fetal SO2 later in gestation. MRI feto-placental SO2 distinguishes between normally grown and FGR fetuses, as well as distinguishing FGR fetuses with and without normal Doppler in humans. Thus, a multi-compartment placental MRI model detects low placental SO2 and distinguishes between small hypoxemic fetuses and normally grown fetuses.

Impact of the Addition of a Centrifugal Pump in a Preterm Miniature Pig Model of the Artificial Placenta.

The recent demonstration of normal development of preterm sheep in an artificial extrauterine environment has renewed interest in artificial placenta (AP) systems as a potential treatment strategy for extremely preterm human infants. However, the feasibility of translating this technology to the human preterm infant remains unknown. Here we report the support of 13 preterm fetal pigs delivered at 102 ± 4 days (d) gestation, weighing 616 ± 139 g with a circuit consisting of an oxygenator and a centrifugal pump, comparing these results with our previously reported pumpless circuit (n = 12; 98 ± 4 days; 743 ± 350 g). The umbilical vessels were cannulated, and fetuses were supported for 46.4 ± 46.8 h using the pumped AP versus 11 ± 13 h on the pumpless AP circuit. Upon initiation of AP support on the pumped system, we observed supraphysiologic circuit flows, tachycardia, and hypertension, while animals maintained on a pumpless AP circuit exhibited subphysiologic flows. On the pumped AP circuit, there was a progressive decline in umbilical vein (UV) flow and oxygen delivery. We conclude that the addition of a centrifugal pump to the AP circuit improves survival of preterm pigs by augmenting UV flow through the reduction of right ventricular afterload. However, we continued to observe the development of heart failure within a matter of days.

Maternal-placental-fetal drug metabolism is altered by late gestation undernutrition in the pregnant ewe.

BACKGROUND: Maternal undernutrition during pregnancy disrupts both fetal growth and development with perturbations to certain physiological processes within the maternal-fetal-placental unit, including metabolic function. However, it is unknown if hypoglycemia during pregnancy alters maternal-fetal-placental drug metabolism as mediated by cytochrome P450 (CYP) enzymes. Despite this, hypoglycemia reduces CYP enzyme activity in non-pregnant animals. We therefore hypothesised that in a sheep model of hypoglycemia induced by late gestation undernutrition (LGUN), maternal-fetal-placental CYP activity would be reduced, and that fetal glucose infusion (LGUN+G) would rescue reduced CYP activity. METHODS: At 115d gestation (term, 150d), ewes were allocated to control (100% metabolic energy requirement (MER); n = 11), LGUN (50% MER; n = 7) or LGUN+G (50% MER + fetal glucose infusion; n = 6) and maintained on their diets until post-mortem. Maternal-fetal-placental CYP activity assays were performed at 131-133d gestation. Microsomes were isolated from placenta and fetal liver collected at 139-142d gestation and incubated with CYP-specific probe drugs. Metabolite concentrations were measured using Liquid Chromatography - tandem mass spectrometry (LC-MS/MS). RESULTS: CYP2C19 and CYP3A were undetectable in placenta or fetal liver, and CYP1A2 was undetectable in the fetal liver. Placental-specific CYP1A2 and CYP2D6 activity and hepatic-specific CYP2D6 activity were unaffected by LGUN. Maternal-fetal-placental CYP1A2 activity was reduced in response to LGUN in the maternal compartment only. CONCLUSIONS: Reduced maternal-fetal-placental CYP1A2 activity, but not placental-specific CYP1A2 activity, may lead to the developing fetus being exposed to increased concentrations of CYP1A2-specific substrates and suggests further consideration of drug dosing is required in instances of late gestation maternal undernutrition.

Development of an optical fiber-based redox monitoring system for tissue metabolism.

An optical redox ratio can potentially be used to report on the dynamics of cell and tissue metabolism and define altered metabolic conditions for different pathologies. While there are methods to measure the optical redox ratio, they are not particularly suited to real-time in situ or in vivo analysis. Here, we have developed a fiber-optic system to measure redox ratios in cells and tissues and two mathematical models to enable real-time, in vivo redox measurements. The optical redox ratios in tissue explants are correlated directly with endogenous NADH/FAD fluorescence emissions. We apply the mathematical models to the two-photon microscopy data and show consistent results. We also used our fiber-optic system to measure redox in different tissues and show consistent results between the two models, hence demonstrating proof-of-principle. This innovative redox monitoring system will have practical applications for defining different metabolic disease states.