Said H. Audi

Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress

Ventilation with enhanced fractions of O(2) (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O(2)) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs.

Accounting for the Heterogeneity of Capillary Transit Times in Modeling Multiple Indicator Dilution Data

AbstractTo mathematically model multiple indicator dilution (MID) data for the purpose of estimating parameters descriptive of indicator-tissue interactions, it is necessary to account for the effects of the distribution of capillary transit times, hc(t) In this paper, we present an efficient approach for incorporating hc(t) in the mathematical modeling of MID data. In this method, the solution of the model partial differential equations obtained at different locations along the model capillary having the longest transit time provides the outflow concentrations for all capillaries. When weighted by hc(t) these capillary outflow concentrations provide the outflow concentration versus time curve for the capillary bed. The method is appropriate whether the available data on capillary dispersion are in terms of capillary transit time or relative flow distributions, and whether the dispersion results from convection time differences among heterogeneous parallel pathways or axial diffusion along individual pathways. Finally, we show that the knowledge of a relationship among the moments of hc(t) rather than hc(t) per se, is sufficient information to account for the effect of hc(t) in the mathematical modeling interpretation of MID data. This relationship can be determined by including a flow-limited indicator in the injected bolus, thus providing an efficient means for obtaining the experimental data sufficient to account for capillary flow and transit time heterogeneity in MID modeling.

Coenzyme Q1 redox metabolism during passage through the rat pulmonary circulation and the effect of hyperoxia

The objective was to evaluate the pulmonary disposition of the ubiquinone homolog coenzyme Q(1) (CoQ(1)) on passage through lungs of normoxic (exposed to room air) and hyperoxic (exposed to 85% O(2) for 48 h) rats. CoQ(1) or its hydroquinone (CoQ(1)H(2)) was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of CoQ(1)H(2) and CoQ(1) were measured. CoQ(1)H(2) appeared in the venous effluent when CoQ(1) was infused, and CoQ(1) appeared when CoQ(1)H(2) was infused. In normoxic lungs, CoQ(1)H(2) efflux rates when CoQ(1) was infused decreased by 58 and 33% in the presence of rotenone (mitochondrial complex I inhibitor) and dicumarol [NAD(P)H-quinone oxidoreductase 1 (NQO1) inhibitor], respectively. Inhibitor studies also revealed that lung CoQ(1)H(2) oxidation was via mitochondrial complex III. In hyperoxic lungs, CoQ(1)H(2) efflux rates when CoQ(1) was infused decreased by 23% compared with normoxic lungs. Based on inhibitor effects and a kinetic model, the effect of hyperoxia could be attributed predominantly to 47% decrease in the capacity of complex I-mediated CoQ(1) reduction, with no change in the other redox processes. Complex I activity in lung homogenates was also lower for hyperoxic than for normoxic lungs. These studies reveal that lung complexes I and III and NQO1 play a dominant role in determining the vascular concentration and redox status of CoQ(1) during passage through the pulmonary circulation, and that exposure to hyperoxia decreases the overall capacity of the lung to reduce CoQ(1) to CoQ(1)H(2) due to a depression in complex I activity.

Pulmonary arterial endothelial cells affect the redox status of coenzyme Q0

The pulmonary endothelium is capable of reducing certain redox-active compounds as they pass from the systemic venous to the arterial circulation. This may have important consequences with regard to the pulmonary and systemic disposition and biochemistry of these compounds. Because quinones comprise an important class of redox-active compounds with a range of physiological, toxicological, and pharmacological activities, the objective of the present study was to determine the fate of a model quinone, coenzyme Q0 (Q), added to the extracellular medium surrounding pulmonary arterial endothelial cells in culture, with particular attention to the effect of the cells on the redox status of Q in the medium. Spectrophotometry, electron paramagnetic resonance (EPR), and high-performance liquid chromatography (HPLC) demonstrated that, when the oxidized form Q is added to the medium surrounding the cells, it is rapidly converted to its quinol form (QH2) with a small concentration of semiquinone (Q*-) also detectable. The isolation of cell plasma membrane proteins revealed an NADH-Q oxidoreductase located on the outer plasma membrane surface, which apparently participates in the reduction process. In addition, once formed the QH2 undergoes a cyanide-sensitive oxidation by the cells. Thus, the actual rate of Q reduction by the cells is greater than the net QH2 output from the cells.

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes.

Our goal was to quantify mitochondrial and plasma potential (Δψ(m) and Δψ(p)) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([R(e)]) were measured over time. R123 [R(e)] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [R(e)] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K(+) concentration ([K(+)]), used to manipulate contributions of membrane potentials, attenuated decreases in [R(e)], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [R(e)]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [R(e)] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψ(m) (-130 ± 7 and -133 ± 4 mV), Δψ(p) (-36 ± 4 and -49 ± 4 mV), and a Pgp activity parameter (K(Pgp), 25 ± 5 and 51 ± 11 μl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψ(m) (-124 ± 6 mV). The consistency of kinetic parameter values obtained from R123 and TMRE data demonstrates the utility of this experimental and theoretical approach for quantifying intact cell Δψ(m) and Δψ(p.) Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O(2) for 48 h) cells have equivalent resting Δψ(m), hyperoxic cell Δψ(m) was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction.

An Interpretation of 14C-Urea and 14C-Primidone Extraction in Isolated Rabbit Lungs

We measured the venous concentration versus time curves of14C-urea and14C-primidone after rapid bolus injections of a vascular reference indicator, fluorescein isothiocyanate dextran, and one of the two14C-labeled indicators in isolated rabbit lungs perfused with Krebs-Ringer bicarbonate solution containing 4.5% bovine serum albumin at flow rates (F) of 6.67, 3.33, 1.67, and 0.83 ml/sec and with nearly constant microvascular pressure and total lung vascular volume. When we calculated the permeability-surface area product,PS, from the14C-urea and14C-primidone outflow curves using the Crone model, the estimates of thePS product were directly proportional toF. However, the fractional change in thePS with flow was different for the two indicators. We also estimated thePS from the same14C-urea and14C-primidone data using an alternative model that includes perfusion heterogeneity, estimated in a previous study, and flow-limited and barrier-limited extravascular volumes accessible to both urea and primidone. This model was able to fit the outflow curves of either14C-urea or14C-primidone at all four flows studied with one flow-independentPS for each indicator. The ability of the new model to explain the14C-urea and14C-primidone data with no flow-dependent change inPS suggests that a change inPS withF estimated using other models such as the Crone model is not sufficient evidence for capillary surface area recruitment.

Differential Lung Uptake of 99mTc-Hexamethylpropyleneamine Oxime and 99mTc-Duramycin in the Chronic Hyperoxia Rat Model

Noninvasive radionuclide imaging has the potential to identify and assess mechanisms involved in particular stages of lung injury that occur with acute respiratory distress syndrome, for example. Lung uptake of 99mTc-hexamethylpropyleneamine oxime (HMPAO) is reported to be partially dependent on the redox status of the lung tissue whereas 99mTc-duramycin, a new marker of cell injury, senses cell death via apoptosis or necrosis. Thus, we investigated changes in lung uptake of these agents in rats exposed to hyperoxia for prolonged periods, a common model of acute lung injury. Methods: Male Sprague–Dawley rats were preexposed to either normoxia (21% O2) or hyperoxia (85% O2) for up to 21 d. For imaging, the rats were anesthetized and injected intravenously with either 99mTc-HMPAO or 99mTc-duramycin (both 37–74 MBq), and planar images were acquired using a high-sensitivity modular γ-camera. Subsequently, 99mTc-macroagreggated albumin (37 MBq, diameter 10–40 μm) was injected intravenously, imaged, and used to define a lung region of interest. The lung-to-background ratio was used as a measure of lung uptake. Results: Hyperoxia exposure resulted in a 74% increase in 99mTc-HMPAO lung uptake, which peaked at 7 d and persisted for the 21 d of exposure. 99mTc-duramycin lung uptake was also maximal at 7 d of exposure but decreased to near control levels by 21 d. The sustained elevation of 99mTc-HMPAO uptake suggests ongoing changes in lung redox status whereas cell death appears to have subsided by 21 d. Conclusion: These results suggest the potential use of 99mTc-HMPAO and 99mTc-duramycin as redox and cell-death imaging biomarkers, respectively, for the in vivo identification and assessment of different stages of lung injury.

The feasibility of imaging myocardial ischemic/reperfusion injury using 99mTc-labeled duramycin in a porcine model

UNLABELLEDnWhen pathologically externalized, phosphatidylethanolamine (PE) is a potential surrogate marker for detecting tissue injuries. (99m)Tc-labeled duramycin is a peptide-based imaging agent that binds PE with high affinity and specificity. The goal of the current study was to investigate the clearance kinetics of (99m)Tc-labeled duramycin in a large animal model (normal pigs) and to assess its uptake in the heart using a pig model of myocardial ischemia-reperfusion injury.nnnMETHODSnThe clearance and distribution of intravenously injected (99m)Tc-duramycin were characterized in sham-operated animals (n=5). In a closed chest model of myocardial ischemia, coronary occlusion was induced by balloon angioplasty (n=9). (99m)Tc-duramycin (10-15mCi) was injected intravenously at 1hour after reperfusion. SPECT/CT was acquired at 1 and 3hours after injection. Cardiac tissues were analyzed for changes associated with acute cellular injuries. Autoradiography and gamma counting were used to determine radioactivity uptake. For the remaining animals, (99m)Tc-tetrafosamin scan was performed on the second day to identify the infarct site.nnnRESULTSnIntravenously injected (99m)Tc-duramycin cleared from circulation predominantly via the renal/urinary tract with an α-phase half-life of 3.6±0.3minutes and β-phase half-life of 179.9±64.7minutes. In control animals, the ratios between normal heart and lung were 1.76±0.21, 1.66±0.22, 1.50±0.20 and 1.75±0.31 at 0.5, 1, 2 and 3hours post-injection, respectively. The ratios between normal heart and liver were 0.88±0.13, 0.80±0.13, 0.82±0.19 and 0.88±0.14. In vivo visualization of focal radioactivity uptake in the ischemic heart was attainable as early as 30min post-injection. The in vivo ischemic-to-normal uptake ratios were 3.57±0.74 and 3.69±0.91 at 1 and 3hours post-injection, respectively. Ischemic-to-lung ratios were 4.89±0.85 and 4.93±0.57; and ischemic-to-liver ratios were 2.05±0.30 to 3.23±0.78. The size of (99m)Tc-duramycin positive myocardium was qualitatively larger than the infarct size delineated by the perfusion defect in (99m)Tc-tetrafosmin uptake. This was consistent with findings from tissue analysis and autoradiography.nnnCONCLUSIONn(99m)Tc-duramycin was demonstrated, in a large animal model, to have suitable clearance and biodistribution profiles for imaging. The agent has an avid target uptake and a fast background clearance. It is appropriate for imaging myocardial injury induced by ischemia/reperfusion.

In vivo detection of hyperoxia-induced pulmonary endothelial cell death using 99mTc-Duramycin

INTRODUCTIONn(99m)Tc-duramycin, DU, is a SPECT biomarker of tissue injury identifying cell death. The objective of this study is to investigate the potential of DU imaging to quantify capillary endothelial cell death in rat lung injury resulting from hyperoxia exposure as a model of acute lung injury.nnnMETHODSnRats were exposed to room air (normoxic) or >98% O2 for 48 or 60 hours. DU was injected i.v. in anesthetized rats, scintigraphy images were acquired at steady-state, and lung DU uptake was quantified from the images. Post-mortem, the lungs were removed for histological studies. Sequential lung sections were immunostained for caspase activation and endothelial and epithelial cells.nnnRESULTSnLung DU uptake increased significantly (p<0.001) by 39% and 146% in 48-hr and 60-hr exposed rats, respectively, compared to normoxic rats. There was strong correlation (r(2)=0.82, p=0.005) between lung DU uptake and the number of cleaved caspase 3 (CC3) positive cells, and endothelial cells accounted for more than 50% of CC3 positive cells in the hyperoxic lungs. Histology revealed preserved lung morphology through 48 hours. By 60 hours there was evidence of edema, and modest neutrophilic infiltrate.nnnCONCLUSIONSnRat lung DU uptake in vivo increased after just 48 hours of >98% O2 exposure, prior to the onset of any substantial evidence of lung injury. These results suggest that apoptotic endothelial cells are the primary contributors to the enhanced DU lung uptake, and support the utility of DU imaging for detecting early endothelial cell death in vivo.

Understanding the in vivo uptake kinetics of a phosphatidylethanolamine-binding agent 99mTc-Duramycin

INTRODUCTIONn(99m)Tc-Duramycin is a peptide-based molecular probe that binds specifically to phosphatidylethanolamine (PE). The goal was to characterize the kinetics of molecular interactions between (99m)Tc-Duramycin and the target tissue.nnnMETHODSnHigh level of accessible PE is induced in cardiac tissues by myocardial ischemia (30xa0min) and reperfusion (120xa0min) in Sprague-Dawley rats. Target binding and biodistribution of (99m)Tc-duramycin were captured using SPECT/CT. To quantify the binding kinetics, the presence of radioactivity in ischemic versus normal cardiac tissues was measured by gamma counting at 3, 10, 20, 60 and 180xa0min after injection. A partially inactivated form of (99m)Tc-Duramycin was analyzed in the same fashion. A compartment model was developed to quantify the uptake kinetics of (99m)Tc-Duramycin in normal and ischemic myocardial tissue.nnnRESULTSn(99m)Tc-duramycin binds avidly to the damaged tissue with a high target-to-background radio. Compartment modeling shows that accessibility of binding sites in myocardial tissue to (99m)Tc-Duramycin is not a limiting factor and the rate constant of target binding in the target tissue is at 2.2xa0ml/nmol/min/g. The number of available binding sites for (99m)Tc-Duramycin in ischemic myocardium was estimated at 0.14xa0nmol/g. Covalent modification of D15 resulted in a 9-fold reduction in binding affinity.nnnCONCLUSIONn(99m)Tc-Duramycin accumulates avidly in target tissues in a PE-dependent fashion. Model results reflect an efficient uptake mechanism, consistent with the low molecular weight of the radiopharmaceutical and the relatively high density of available binding sites. These data help better define the imaging utilities of (99m)Tc-Duramycin as a novel PE-binding agent.