Non-invasive monitoring of mitochondrial oxygenation and respiration in critical illness using a novel technique

The background of the PpIX-TSLT is described in detail elsewhere [20‐22, 24]. In short, PpIX is the final precursor of heme in the heme biosynthetic pathway. PpIX is synthesized in the mitochondria, and administration of ALA substantially enhances the PpIX concentration. Since the conversion of PpIX to heme is a rate-limiting step, administration of ALA causes accumulation of PpIX inside the mitochondria (Fig. 1). PpIX possesses a triplet state that reacts strongly with oxygen, making its lifetime oxygen dependent. Population of the first excited triplet state occurs upon photoexcitation with a pulse of light, and causes the emission of red delayed fluorescence. The delayed fluorescence lifetime is related to the mitoPO₂ according to the Stern–Volmer equation:

in which τ is the measured delayed fluorescence lifetime, k_q is the quenching constant, and τ₀ is the lifetime at zero oxygen. In the case of a nonhomogeneous oxygen distribution inside the measurement volume, a reliable estimation of the average PO₂ can be made by the rectangular distribution method (RDM) [27, 28].

A compact computer-controlled tunable laser (Opolette 355-I; Opotek, Carlsbad, CA, USA), providing pulses with a specified duration of 4–10 nanoseconds and typically 2–4 mJ/pulse over the tunable range of 410–670 nm, was used as excitation source. The laser was coupled into a Fiber Delivery System (Opotek) consisting of a 50 mm planoconvex lens, an X–Y fiber mount, and a 2 m fiber with a core diameter of 1000 μm. This fiber was coupled to a custom-made reflection probe by an In-Line Fiber Optic Attenuator (FOA-Inline; Avantes b.v., Eerbeek, the Netherlands). The reflection probe consisted of two 1000 μm fibers with a length of 2 m (P1000-2-VIS-NIR; Ocean Optics, Dunedin, FL, USA) mounted at the common end into a stainless steel holder with a separation of 1 mm between the fibers. The common end of the reflection probe consisted of an aluminum rod with a length of 5 cm and a diameter of 10 mm. The light output of the excitation branch was measured by a FieldMate laser power meter with a PowerMax PS19 measuring head (Coherent Inc., Santa Clara, CA, USA). Our experiments used an excitation energy of 250 μJ/pulse and a repetition frequency of 1 Hz. The PpIX signal was detected by a gated microchannel plate photomultiplier tube (MCP-PMT R5916U series; Hamamatsu Photonics, Hamamatsu, Japan). The emission branch of the reflection probe was fit into an Oriel Fiber Bundle Focusing Assembly (Model 77799; Newport, Irvine, CA, USA) which was coupled to the MCP-PMT by an in-house built optics consisting of a filter-holder, a plano convex lens (BK-7; OptoSigma, Santa Ana, CA, USA) with focal length of 90 mm, and an electronic shutter (04 UTS 203; Melles Griot, Albuquerque, NM, USA). The PpIX emission light was filtered by a combination of a 590 nm longpass filter (OG590; Newport) and a broadband (675–25 nm) bandpass filter (Omega Optical, Brattleboro, VT, USA).

The local oxygen consumption is measured by the ODR after local cessation of the oxygen supply by pressure-induced occlusion of the microcirculation. The reflection probe was mounted above the ALA cream-treated skin with a height-adjustable stand, allowing different settings for the probe-to-skin distance. Occlusion of the microcirculation in the skin was obtained by local pressure with the measurement probe. This simple procedure reliably created stop-flow conditions and induced a measurable ODR, due to cessation of the microvascular oxygen supply and ongoing cellular oxygen consumption. The mitoPO₂ was measured before and during application of pressure at an interval of 1 Hz, using one laser pulse per measurement. The principles of ODR measurements are shown in Fig. 2. We have described these principles in detail and provided a working implementation of the technique for ODR measurements previously [26]. In that implementation, we developed a method for the analysis of delayed-fluorescence-based ODR data to calculate the mitoPO₂, ODR, and P50 and demonstrated the feasibility and reproducibility of our method in rats [26].

The protocol was approved by the Animal Research Committee of the Erasmus University Medical Center Rotterdam, the Netherlands. Animal care and handling were performed in accordance with the guidelines for Institutional and Animal Care and Use Committees.

A total of 28 male Wistar rats (mean body weight 292 g, standard deviation ± 25.5 g) were used in this study. All animals were anesthetized by intraperitoneal injection of a mixture of ketamine 90 mg/kg (Alfasan, Woerden, the Netherlands), 0.5 mg/kg medetomidine (Sedator Eurovet Animal Health BV, Bladel, the Netherlands), and 0.05 mg/kg atropine (Centrofarm Services BV, Etten-Leur, the Netherlands). Ketamine (50 mg/kg/hour) and crystalloid solution (Ringer’s lactate, 5 ml/kg/hour) were infused intravenously to maintain anesthesia and fluid balance. Following tracheotomy, mechanical ventilation was instigated (Babylog 8000 plus; Dräger, Lubeck, Germany). Ventilation was adjusted on end-tidal carbon dioxide tension, keeping the arterial carbon dioxide tension at 35–45 mmHg. The inspired oxygen concentration was set at 40 %. A polyethylene catheter (outer diameter 0.9 mm) was inserted into the right jugular vein for intravenous administration of a colloid solution (Voluven 3.5 ml/kg/hour). Arterial blood pressure and heart rate were monitored with a similar catheter in the left femoral artery. Every hour we performed analysis of arterial blood gas, and measured cardiac output by thermodilution (PowerLab; ADInstruments, Colorado Springs, CO, USA) with the thermistor inserted in the right carotid artery. Body temperature was rectally measured and maintained at 38 ± 0.5 °C by means of a heating pad.

In all groups PpIX was induced by topical application of 2.5 % ALA cream. The ALA cream was prepared by mixing hydrophilic cremor lanette (Lanettecreme I FNA; Bipharma, Weesp, the Netherlands) and 2.5 % ALA (Sigma-Aldrich, St. Louis, MO, USA) just before use. The cream was topically administered on the abdominal skin after hair removal. The latter was accomplished by shaving and subsequent use of hair removal cream (Veet; Reckitt Benckiser Co., Slough, UK). The ALA-treated skin was covered with an adhesive film to avoid evaporation. Furthermore, to prevent premature exposure of the primed skin to light, the primed area was covered with aluminum foil. After 3 hours, sufficient PpIX had accumulated to commence baseline (t0) in vivo measurements of the mitoPO₂ and ODR. All mitoPO₂ and ODR measurements were performed in a dark environment to prevent ambient light contamination.

After measurements at t0, animals were assigned to one of three groups: lipopolysaccharide without fluid resuscitation (LPS-NR, n = 10), LPS with fluid resuscitation (LPS-FR, n = 10), and untreated control (n = 8). The LPS groups received an intravenous LPS injection (1.6 mg/kg during 10 minutes) (extracted from Escherichia coli, serotype 0127:B8; Sigma-Aldrich). The LPS was dissolved at a concentration of 1 mg/ml in a crystalloid solution. The oxygen measurements were repeated 3 h after infusion of LPS (t1).

In the LPS-FR group, fluid resuscitation was achieved by continuous infusion of a colloid solution at 7 ml/kg/hour (Voluven; Fresenius Kabi, Bad Homburg, Germany); a 2 ml bolus of the same colloid solution was administered just before the start of the t2 measurements. Finally, in the LPS-NR group a 2 ml colloid fluid bolus (Voluven; Fresenius Kabi) was given directly after t1 measurements to achieve a late resuscitation model (LPS-LR). The effect of this late resuscitation on the mitoPO₂ and ODR was measured directly after fluid administration (t2). Figure 3 describes the experimental setup.

Statistical analyses were performed using IBM SPSS version 21 (IBM Corporation, Armonk, NY, USA). Unless stated otherwise, all values are reported as mean ± standard error.

Statistical significance between groups was calculated using a two-tailed Student's t test. A paired Student’s t test was used to compare differences between measurements at t0 and t1. p ≤0.05 was considered statistically significant.