Evaluation of endoscopic visible light spectroscopy: comparison with microvascular oxygen tension measurements in a porcine model

This study was approved by the local Animal Research Committee of the Erasmus MC University Medical Center in accordance with the National Guidelines for Animal Care and Handling (protocol number DEC 129-13-06 EMC3185). To enhance transparency this article is written according to the ARRIVE guidelines for animal research [19].

In total, 5 female crossbred Landrace x Yorkshire pigs, with mean body weights of 28.1 ± 0.6 kg (mean ± standard error of mean), age 2–3 months were used for the experiments. Sample size calculation determined that 5 animals were sufficient to detect a difference of at least 5% in mucosal saturation measured with VLS before and after bile per location with an alpha of 0.05 and a power of 90% [20].

After an overnight fast with free access to water, the animals were sedated with an intramuscular injection of tiletamine/zolazepam (6/6 mg/kg; Virbac Laboratories, Carros, France), xylazine (2 mg/kg; AST Farma B.V., The Netherlands) and atropine sulfate (0.5 mg/animal; Centrafarm Services BV, Etten-Leur, The Netherlands). After a 15 min induction period, anesthesia was induced with tilatamine/zolazepam (50–100 mg/animal) through a cannula (20G Venflon (Becton, Dickinson and Company, USA) in an auricular vein. Tracheal intubation was performed with a size 7.0 Portex^® endotracheal tube (Smiths Medical International Ltd., United Kingdom). For maintenance of anesthesia, the animals received continuous infusion of ketamine (5 mg kg⁻¹ h⁻¹; Alfasan Nederland B.V., The Netherlands), midazolam (1.5 mg kg⁻¹ h⁻¹; Atavis Group PCT, Iceland), sufentanil (4 μg kg⁻¹ h⁻¹; Janssen-Cilag B.V., The Netherlands), and rocuroniumbromide (4 mg kg⁻¹ h⁻¹; Fresenius Kabi Austria GmbH, Austria). All animals received 500 ml of colloid solution (Voluven^®; Fresenius Kabi AG, Germany) at start and a continuous infusion of crystalloid (Sterofundin^® ISO 10 ml kg⁻¹ h⁻¹; B. Braun, Germany). Each pig received a bolus of magnesium sulfate (500 mg; Pharmachemie BV, Haarlem, The Netherlands), as arrhythmia prophylaxis, added to the first bag of crystalloid solution. To prevent infections during the experiment, Cefazolin (1000 mg/animal; Kefzol ^® EuroCept BV, Ankeveen, The Netherlands), an antibiotic used for the treatment of a widespread of bacteria was given intravenous.

Pressure-controlled mechanical ventilation (Servo 300; Siemens-Elema, Solna, Sweden) was performed with a FiO₂ between 24% and a positive end-expiratory pressure of 5 cm H₂O while no intervention was done. Normothermia, measured nasal, was maintained between 38 and 39 °C, with two heating pads underneath and an electric heating blanket above the animal. Furthermore, hearth rate, MAP, SpO₂ and temperature were monitored continuously throughout the entire experiment. Arterial blood samples were collected to determine the arterial oxygen pressure and arterial oxygen saturation (ABL 800Flex (Radiometer, Denmark).

A 4F thermodilution catheter (Pulsion Medical Systems AG München, Germany) was placed in the left femoral artery for arterial blood sampling. An 9Fr introducer sheath (Arrow International Inc., USA) was placed in the right jugular vein for infusion of palladium porphyrin. Both catheters were placed using the Seldinger technique. A lower midline abdominal incision was made to insert a cystostomy tube into the urinary bladder with purse-string sutures for urine collection.

The animals were placed in supine position and an incision was made to open the abdomen. A small intestinal loop was dissected and a small incision was made at the non-vascularized side to expose the intestinal mucosa (Fig. 1). Mucosal oxygen saturation measurements were performed with a fiberoptic probe (Endoscopic T-Stat Sensor; Spectros, Portola Valley, California, USA) connected to the VLS oximeter (T-Stat 303 Microvascular Oximeter, Spectros, Portola Valley, California).

Microvascular oxygen tension measurements were done with oxygen dependent phosphorescent dye palladium porphine (Pd-porphyrin). Palladium porphyrin is a large molecule with optical properties that can absorb energy and react with oxygen. In the absence of oxygen it will release the absorbed energy from an excitation source via phosphorescent light with a specific decay time, i.e. lifetime. The lifetime is related to the amount of oxygen surrounding the Pd-porphyrin described by the Stern–Volmer relation [18]. It has been tested for pH, temperature and diffusivity dependency [17]. Calibration experiments are done and determine the O₂ accuracy of 5% independent of phosphorescence intensity itself [17].

For the laboratory experimental setup of the μPO₂ measurements the excitation source was an Opolette 355-I tunable laser (Opotek, Carlsbad, CA, USA) set to a wavelength of 524 nm. An optical fiber developed by TNO and produced by Light Guide Optics was used that would fit through the working channel of a gastroduodenal endoscope. It has one central located excitation fiber with several surrounding detection fibers.

The phosphorescence was collected with a gated micro channel plate photomultiplier tube (MCP-PMT R5916U series, Hamamatsu Photonics, Hamamatsu, Japan). Phosphorescence lifetime analysis was done with a self-written software program in Labview (version 13.0, National Instruments, Austin, TX, USA). For the detailed setup description we refer elsewhere [21].

The probe palladium porphyrin was Pd(II) meso-Tetra (4-carboxyphenyl)porphine (80 mg/animal) (Frontier Scientific, Logan, USA) dissolved in 1 ml DMSO and TRIS Trisma^® Base (Sigma, St. Louis, MO) was combined with a 4% bovine serum albumin solution solved in phosphate buffered saline. This method has been validated in vitro and in vivo [17]. Pd-porphyrin bound to albumin, forms a high-molecular-weight complex, confining it mainly to the vascular compartment when infused intravenously.

Both optical fibers were fixated together to perform stable simultaneous mucosal oxygen saturation and μPO₂ measurements of the same mucosal spot of the small intestine (Fig. 1).

Simultaneous VLS mucosal oxygen saturation and μPO₂ measurements were performed at different FiO₂ values ranging from 18 to 100%. The mucosal oxygen saturation and μPO₂ measurements were simultaneously performed for 2 min at a specific FiO₂ value. When a new FiO₂ value was set, the start of a set of new measurements was awaited for the first two 2 min. To compare the two measurement techniques the μPO₂ was converted into a corresponding saturation. For the μPO₂ conversion, for every measured value in mmHg the corresponding % was calculated called micro-vascular oxygen saturation converted (μSO₂.converted). The conversion can be found in Fig. 2.

Furthermore, the influence of bile on mucosal oxygen saturation values measured with VLS was assessed. Mucosal oxygen saturation measurements were performed of the small intestine mucosa in presence of bile. Two different types of bile were used: fluid obtained during upper GI endoscopy from the stomach of the animal and fluid obtained from the small intestine of the animal. The sticky viscosity of the bile ensured the fixation of the bile on the measurement area and continuous visual confirmation ensured that the bile measurements were performed on surface covered with bile. The amount of bile applied to the mucosa, the thickness of the bile applied and the exact content of the bile applied were not controlled. The mucosal oxygen saturations in presence of bile were compared with the mucosal oxygen saturations before the bile was applied to the mucosa (baseline) and the mucosal oxygen saturations every time after the bile was removed with saline fluid as control. For every step approximately 30 measurements were done.

Finally, simultaneous mucosal oxygen saturation and μPO₂ measurements were performed from the moment a lethal dose potassium chloride was intravenously injected. A measurement period of 25 min after injection was considered long enough to ensure a steady state since Benaron et al. showed detection of local ischemia with VLS within 120 s [22].

Mucosal oxygen saturation values were defined in percentage tissue hemoglobin saturation. The μPO₂ measurements were defined in mmHg.

Statistical analysis was performed with R Statistics software (v3.2.4). Normal distribution was assessed visually and with the Shapiro–Wilk normality test. Normal distributed data is presented as mean ± standard deviation (SD) and abnormally distributed data is presented as median with interquartile range (IQR). A linear regression model was used for the FiO₂, mucosal oxygen saturations, and μPO₂. A scatter plot was used to show the mucosal oxygen saturation versus the μPO₂ measurements at different FiO₂ values. To compare the two measurement techniques, the μPO₂ was converted from mmHg to % porcine hemoglobin saturation. To determine the saturation a porcine-specific hemoglobin saturation formula published by Serianni et al. was used [23]: (%/100) = (0.13534 × P_O2)^3.02/[(0.13534 − P_O2)^3.02 + 91.2]. The formula was derived from 213 data point at pH 7.4 and 37° with an excellent fit.

To compare the saturation, the difference in measurement frequency had to be overcome. The mucosal oxygen saturation has a fixed measurement interval whereas the μPO₂ is measured on demand. To equally compare the two measurements the mucosal oxygen saturation was averaged over same period as one μPO₂ was done. Thereafter these results were visualized with linear regression and with a Bland–Altman comparison plot [24].

The Wilcoxon signed-rank test was used to compare the measurement before, with and after application of bile. A two-tailed p value of < 0.05 was considered significant. After the potassium chloride injection mucosal oxygen saturation measurements were compared with μPO₂. Because VLS measures every second, a symmetrical moving average of 20 samples was taken to smooth the data, for example the eleventh sample is an average of sample [1‐21].

All 5 animals were in good clinical condition before the start of the experiment.

The mucosal oxygen saturation levels versus the μPO₂ levels different values of FiO₂ in 5 animals were measured. The mucosal oxygen saturation decreased with increasing FiO₂ in contrast to the μPO₂ values that increased with increasing FiO₂. The spread of the mucosal oxygen saturation levels and the FiO₂ levels was large, shown in Fig. 3.

Figure 4a shows the correlation between mucosal oxygen saturation and the converted μPO₂ saturation. There is a poor linear correlation with an r² = 0.39, an interception of 18.5% and a slope of 0.41. In the Bland–Altman plot (Fig. 4b) also a poor correlation is seen with a mean difference of − 16%. If the saturation increases the mucosal oxygen saturation undervalues the saturation even more.

Figure 5 shows the mucosal oxygen saturation measurements without the presence of bile, with the presence of a bile mixture from the stomach and with the presence of a bile mixture from the small bowel and measurements without any of the bile mixtures measured in a total of 2 animals. The mucosal oxygen saturation measurements before application of the bile mixtures and after the bile mixtures were removed were not significantly different (mucosal oxygen saturation before application of bile mixture median (IQR) 57.5% (54.8–59.0%) versus mucosal oxygen saturation after removal bile mixture 57.0% (54.7–58.6%), p = 0.2743). However, a significant increase of the mucosal oxygen saturation was seen when the bile mixture from the stomach was applied compared to the mucosal oxygen saturation before application of the bile mixtures (median mucosal oxygen saturation with mixture of the stomach (IQR) 73.5% (66.8–85.8) p = < 2.2 * 10⁻¹⁶). When the bile mixture from the small bowel was applied, the mucosal oxygen saturation was significantly lower with a median (IQR) 47.6% (41.8–50.8), p = < 2.2 * 10⁻¹⁶ compared to mucosal oxygen saturation measurements with bile mixture form the stomach and the mucosal oxygen saturation increased significantly after the bile mixtures had been removed (p = < 2.2 * 10⁻¹⁶).

The mucosal oxygen saturation measurements and μPO₂ measurements during the minimally first 25 min of asystole in 5 animals are shown in Fig. 6. In all 5 animals the μPO₂ measurements decreased towards a value of 0. The mucosal oxygen saturation measured with VLS decreased and increased variably during the measurement period and the mucosal oxygen saturation never reached a stable state around 0%.

No adverse events occurred during the 5 porcine experiments.