Egbert G. Mik

Microvascular and mitochondrial PO 2 simultaneously measured by oxygen-dependent delayed luminescence

Measurement of tissue oxygenation is a complex task and various techniques have led to a wide range of tissue PO(2) values and contradictory results. Tissue is compartmentalized in microcirculation, interstitium and intracellular space and current techniques are biased towards a certain compartment. Simultaneous oxygen measurements in various compartments might be of great benefit for our understanding of determinants of tissue oxygenation. Here we report simultaneous measurement of microvascular PO(2) (μPO(2) ) and mitochondrial PO(2) (mitoPO(2) ) in rats. The μPO(2) measurements are based on oxygen-dependent quenching of phosphorescence of the near-infrared phosphor Oxyphor G2. The mitoPO(2) measurements are based on oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Favorable spectral properties of these porphyrins allow simultaneous measurement of the delayed luminescence lifetimes. A dedicated fiber-based time-domain setup consisting of a tunable pulsed laser, 2 red-sensitive gated photomultiplier tubes and a simultaneous sampling data-acquisition system is described in detail. The absence of cross talk between the channels is shown and the feasibility of simultaneous μPO(2) and mitoPO(2) measurements is demonstrated in rat liver in vivo. It is anticipated that this novel approach will greatly contribute to our understanding of tissue oxygenation in physiological and pathological circumstances.

Microcirculatory and mitochondrial hypoxia in sepsis, shock, and resuscitation

After shock, persistent oxygen extraction deficit despite the apparent adequate recovery of systemic hemodynamic and oxygen-derived variables has been a source of uncertainty and controversy. Dysfunction of oxygen transport pathways during intensive care underlies the sequelae that lead to organ failure, and the limitations of techniques used to measure tissue oxygenation in vivo have contributed to the lack of progress in this area. Novel techniques have provided detailed quantitative insight into the determinants of microcirculatory and mitochondrial oxygenation. These techniques, which are based on the oxygen-dependent quenching of phosphorescence or delayed luminescence are briefly reviewed. The application of these techniques to animal models of shock and resuscitation revealed the heterogeneous nature of oxygen distributions and the alterations in oxygen distribution in the microcirculation and in mitochondria. These studies identified functional shunting in the microcirculation as an underlying cause of oxygen extraction deficit observed in states of shock and resuscitation. The translation of these concepts to the bedside has been enabled by our development and clinical introduction of hand-held microscopy. This tool facilitates the direct observation of the microcirculation and its alterations at the bedside under the conditions of shock and resuscitation. Studies identified loss of coherence between the macrocirculation and the microcirculation, in which resuscitation successfully restored systemic circulation but did not alleviate microcirculatory perfusion alterations. Various mechanisms responsible for these alterations underlie the loss of hemodynamic coherence during unsuccessful resuscitation procedures. Therapeutic resolution of persistent heterogeneous microcirculatory alterations is expected to improve outcomes in critically ill patients.

Effects of 1400W and/or nitroglycerin on renal oxygenation and kidney function during endotoxaemia in anaesthetized rats

1 The pathogenesis of acute renal failure (ARF) in sepsis is multifactorial. The role of nitric oxide (NO) in septic ARF has been a source of controversy. We hypothesized that endotoxaemia‐induced exacerbation of inducible nitric oxide synthase (iNOS)‐related NO release impairs renal oxygenation and contributes to ARF in anaesthetized rats. 2 In the present study, rats received lipopolysaccharide (2.5 mg/kg) for 30 min. Two hours later, fluid resuscitation was started (HES130; 5 mL/kg per h after a 5 mL/kg bolus) supplemented either by the NO donor nitroglycerin (NTG; 0.5 µg/kg per min after a 2 µg/kg bolus), the selective iNOS inhibitor 1400W (3 mg/kg per h after a 3 mg/kg bolus) or both. Systemic haemodynamics and renal microvascular Po2 (µPo2) were recorded continuously. Furthermore, creatinine clearance, plasma NOx (nitrate + nitrite + S‐nitrosothiols) levels and the expression of iNOS mRNA were measured. 3 Endotoxaemia reduced renal blood flow, decreased mean arterial pressure, resulted in anuria and was associated with an increase in plasma NOx levels and renal iNOS expression. Renal µPo2 deteriorated gradually during endotoxaemia and there was a significant decrease in renal O2 delivery and consumption. Manipulation of NO levels had no beneficial effect on systemic haemodynamics, renal µPo2 or creatinine clearance over standard fluid resuscitation. The application of 1400W+NTG significantly reduced plasma NOx levels compared with fluid resuscitation and NTG alone. 4 Neither iNOS inhibition, NO donation nor a combination of both showed beneficial effects on systemic haemodynamics, renal oxygenation and renal function compared with fluid resuscitation alone. Our results question the proposed key role of NO in the pathogenesis of septic ARF in rats.

Cutaneous respirometry by dynamic measurement of mitochondrial oxygen tension for monitoring mitochondrial function in vivo

Progress in diagnosis and treatment of mitochondrial dysfunction in chronic and acute disease could greatly benefit from techniques for monitoring of mitochondrial function in vivo. In this study we demonstrate the feasibility of in vivo respirometry in skin. Mitochondrial oxygen measurements by means of oxygen-dependent delayed fluorescence of protoporphyrin IX are shown to provide a robust basis for measurement of local oxygen disappearance rate (ODR). The fundamental principles behind the technology are described, together with an analysis method for retrievel of respirometry data. The feasibility and reproducibility of this clinically useful approach are demonstrated in a series of rats.

Validation of the protoporphyrin IX–triplet state lifetime technique for mitochondrial oxygen measurements in the skin

Mitochondrial oxygen tension can be measured in vivo by means of oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Here we demonstrate that mitochondrial PO(2) (mitoPO(2)) can be measured in the skin of a rat after topical application of the PpIX precursor 5-aminolevulinic acid (ALA). Calibration of mitoPO(2) measurements was done by comparison with simultaneous measurements of the cutaneous microvascular PO(2) This was done under three different conditions: in normal skin tissue, in nonrespiration skin tissue due to the application of cyanide, and in anoxic skin tissue after the ventilation with 100% nitrogen. The results of this study show that it is feasible to measure the mitoPO(2) after the topical application of ALA cream by means of the PpIX-triplet state lifetime technique.

Increased in vivo mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: mitochondrial inhibition as driver of cardiac failure?

BackgroundThe leading cause of mortality due to pulmonary arterial hypertension (PAH) is failure of the cardiac right ventricle. It has long been hypothesized that during the development of chronic cardiac failure the heart becomes energy deprived, possibly due to shortage of oxygen at the level of cardiomyocyte mitochondria. However, direct evaluation of oxygen tension levels within the in vivo right ventricle during PAH is currently lacking. Here we directly evaluated this hypothesis by using a recently reported technique of oxygen-dependent quenching of delayed fluorescence of mitochondrial protoprophyrin IX, to determine the distribution of mitochondrial oxygen tension (mitoPO2) within the right ventricle (RV) subjected to progressive PAH.MethodsPAH was induced through a single injection of monocrotaline (MCT). Control (saline-injected), compensated RV hypertrophy (30xa0mg/kg MCT; MCT30), and RV failure (60xa0mg/kg MCT; MCT60) rats were compared 4 wk after treatment. The distribution of mitoPO2 within the RV was determined in mechanically-ventilated, anaesthetized animals, applying different inspired oxygen (FiO2) levels and two increment dosages of dobutamine.ResultsMCT60 resulted in RV failure (increased mortality, weight loss, increased lung weight), MCT30 resulted in compensated RV hypertrophy. At 30% or 40% FiO2, necessary to obtain physiological arterial PO2 in the diseased animals, RV failure rats had significantly less mitochondria (15% of total mitochondria) in the 0-20xa0mmHg mitoPO2 range than hypertrophied RV rats (48%) or control rats (54%). Only when oxygen supply was reduced to 21% FiO2, resulting in low arterial PO2 for the MCT60 animals, or when oxygen demand increased with high dose dobutamine, the number of failing RV mitochondria with low oxygen became similar to control RV. In addition, metabolic enzyme analysis revealed similar mitochondrial mass, increased glycolytic hexokinase activity following MCT, with increased lactate dehydrogenase activity only in compensated hypertrophied RV.ConclusionsOur novel observation of increased mitochondrial oxygenation suggests down-regulation of in vivo mitochondrial oxygen consumption, in the absence of hypoxia, with transition towards right ventricular failure induced by pulmonary arterial hypertension.

Pathophysiological Consequences of TAT-HKII Peptide Administration Are Independent of Impaired Vascular Function and Ensuing Ischemia

Rationale: We have shown that partial dissociation of hexokinase II (HKII) from mitochondria in the intact heart using low-dose transactivating transcriptional factor (TAT)-HKII (200 nmol/L) prevents the cardioprotective effects of ischemic preconditioning, whereas high-dose TAT-HKII (10 &mgr;mol/L) administration results in rapid myocardial dysfunction, mitochondrial depolarization, and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely because of vasoconstriction and ensuing ischemia. Objective: To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium. Methods and Results: Using a variety of complementary techniques, including mitochondrial membrane potential (&Dgr;&psgr;m) imaging, high-resolution optical action potential mapping, analysis of lactate production, nicotinamide adenine dinucleotide epifluorescence, lactate dehydrogenase release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of ischemic preconditioning, is not associated with ischemia or ischemic injury. Conclusions: Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia, thereby lending further credence to the role of mitochondria-bound HKII as a critical regulator of cardiac function, ischemia-reperfusion injury, and cardioprotection by ischemic preconditioning.

Cutaneous mitochondrial respirometry: non-invasive monitoring of mitochondrial function.

The recently developed technique for measuring cutaneous mitochondrial oxygen tension (mitoPO2) by means of the Protoporphyrin IX—Triplet State Lifetime Technique (PpIX-TSLT) provides new opportunities for assessing mitochondrial function in vivo. The aims of this work were to study whether cutaneous mitochondrial measurements reflect mitochondrial status in other parts of the body and to demonstrate the feasibility of the technique for potential clinical use. The first part of this paper demonstrates a correlation between alterations in mitochondrial parameters in skin and other tissues during endotoxemia. Experiments were performed in rats in which mitochondrial dysfunction was induced by a lipopolysaccharide-induced sepsis (nxa0=xa05) and a time control group (nxa0=xa05). MitoPO2 and mitochondrial oxygen consumption (mitoVO2) were measured using PpIX-TSLT in skin, liver and buccal mucosa of the mouth. Both skin and buccal mucosa show a significant mitoPO2-independent decrease (Pxa0<xa00.05) in mitoVO2 after LPS infusion (a decrease of 37 and 39xa0% respectively). In liver both mitoPO2 and mitoVO2 decreased significantly (33 and 27xa0% respectively). The second part of this paper describes the clinical concept of monitoring cutaneous mitochondrial respiration in man. A first prototype of a clinical PpIX-TSLT monitor is described and its usability is demonstrated on human skin. We expect that clinical implementation of this device will greatly contribute to our understanding of mitochondrial oxygenation and oxygen metabolism in perioperative medicine and in critical illness. Our ultimate goal is to develop a clinical monitor for mitochondrial function and the current results are an important step forward.

Optimizing working space in laparoscopy: CT-measurement of the effect of neuromuscular blockade and its reversal in a porcine model

ObjectiveThe objective of this paper was to determine the effect of neuromuscular blockade (NMB) on working space in a porcine laparoscopy model.BackgroundConflicting results on the effect of NMB on laparoscopic working space are found in literature. Almost all studies are limited by absence of objective assessment of working space or use surrogate outcomes.MethodsIn a standardized porcine laparoscopy model, laparoscopic working-space dimensions with and without NMB were investigated in 16 animals using computed tomography at intra-abdominal pressures of 0, 5, 10, and 15xa0mmHg during multiple runs of abdominal insufflation.ResultsNo statistically significant effect of NMB on abdominal dimensions and laparoscopic working-space volume was found during CO2 pneumoperitoneum. In contrast, the effect of pre-stretching of the abdominal wall by a previous abdominal insufflation was found to be significant.ConclusionsThis experimental study confirms the results from several clinical studies that NMB does not influence laparoscopic working space. Studies dealing with working space during laparoscopy should take note of pre-stretching bias.

A monitor for Cellular Oxygen METabolism (COMET): monitoring tissue oxygenation at the mitochondrial level.

After introduction of the protoporphyrin IX-triplet state lifetime technique as a new method to measure mitochondrial oxygen tension in vivo, the development of a clinical monitor was started. This monitor is the “COMET”, an acronym for Cellular Oxygen METabolism. The COMET is a non-invasive electrically powered optical device that allows measurements on the skin. The COMET is easy to transport, due to its lightweight and compact size. After 5-aminolevulinic acid application on the human skin, a biocompatible sensor enables detection of PpIX in the mitochondria. PpIX acts as a mitochondrially located oxygen-sensitive dye. Three measurement types are available in the touchscreen-integrated user interface, ‘Single’, ‘Interval’ and ‘Dynamic measurement’. COMET is currently used in several clinical studies in our institution. In this first description of the COMET device we show an incidental finding during neurosurgery. To treat persisting intraoperative hypertension a patient was administered clonidine, but due to rapid administration an initial phase of peripheral vasoconstriction occurred. Microvascular flow and velocity parameters measured with laser-doppler (O2C, LEA Medizintechnik) decreased by 44 and 16% respectively, but not the venous-capillary oxygen saturation. However, mitochondrial oxygen tension in the skin detected by COMET decreased from a steady state of 48 to 16xa0mmHg along with the decrease in flow and velocity. We conclude that COMET is ready for clinical application and we see the future for this bedside monitor on the intensive care, operating theater, and testing of mitochondrial effect of pharmaceuticals.