Study design, patient information
This observational study was conducted in accordance with the declaration of Helsinki and after approval by the institutional review board (IRB) of the University of Bonn (Protocol Number 360/16, date of approval March 21, 2018). Patients being scheduled for laparotomy for advanced-stage ovarian or uterine cancer were prospectively screened to participate in the study. Exclusion criteria were as follows: inability or refusal to provide written informed consent, patient age < 18 years, and pregnancy. Prior to anesthesia induction, a thoracic epidural catheter was usually placed for postoperative analgesia. All patients received anesthesia induction according to standard procedures including intubation, femoral arterial line, central venous catheterization, and urinary catheter. Anesthesia was induced with sufentanil, propofol, and rocuronium and was maintained with either isoflurane or sevoflurane and by continuous infusion of remifentanil. In addition to standard monitoring, advanced hemodynamic monitoring was performed using the VolumeView™ system (EV-1000, Edwards Lifesciences Corp., Irvine, CA, USA). Cardiac index (CI), stroke volume index (SVI), stroke volume variation (SVV), and systemic vascular resistance index (SVRI) were measured continuously by pulse contour analysis, while global enddiastolic volume index (GEDI), intrathoracic blood volume index (ITBI), and extravascular lung water index (ELWI) were assessed discontinuously upon transpulmonary thermodilution. Baseline values were determined before beginning of surgery, and intraoperative values were normalized to the baseline.
Intraoperative hemodynamic management followed a goal-directed algorithm. Crystalloid and colloid fluids were administered to keep CI > 3.0 l/min*m2, SVI > 40 mL/m2, and SVV < 15%. When CI and SVI were still below target values despite prior fluid optimization, dobutamine was administered additionally. Norepinephrine was administered in case of a mean arterial pressure (MAP) still < 65 mmHg despite prior fluid optimization. Red blood cell and platelet concentrates and Fresh Frozen Plasma were substituted according to recent transfusion guidelines. If intraoperative determination of serum albumin revealed critically low values (< 20 g/l), human albumin solution was substituted on the responsibility of the attending anesthetist that was not part of the study team. Patients that were administered albumin during surgery were excluded from subsequent analysis of total serum protein.
Depending on the progress of cancer, some patients were treated with hyperthermic intraperitoneal chemotherapy. Upon completion of the surgical procedure, anesthesia was terminated, and patients were extubated if a stable respiratory situation was provided. Subsequently, patients were transferred to the ICU for postoperative care.
Assessment of total serum protein and angiopoietin and soluble TIE2 (sTIE2) levels
Ten mls of blood were drawn before the beginning of surgery (at baseline), intraoperatively every 5000 mL of administered (crystalloid and colloid) infusion solution, and postoperatively. Coagulated samples were centrifuged (3000 rpm, 4 °C, 10 min), and serum aliquots were stored at − 80 °C for subsequent analysis.
Total protein levels were assessed in serum samples using the Pierce™ bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions and as described in [12]. Angiopoietin 1 (ANG-1) was measured using a commercially available ELISA kit (RnD Systems, Minneapolis, MN, USA). ANG-2 and sTIE2 levels were detected using custom-made Luminex™ multiplex arrays purchased from RnD Systems according to the manufacturer’s protocol. Bead-based multiplex arrays such as the Luminex™ system are described in [13]. Arrays were analyzed on a MAGPIX™ reader (Luminex Corp., Austin, TX, USA). Results are given in ng/mL serum. All experimental analyses were performed in duplicates. Out of these results, the mean value was calculated and used for further statistical analyses. All personnel performing the serum analyses were blinded for the intra- and postoperative patient data.
Assessment of endothelial nitric oxide synthase (eNOS) phosphorylation in human dermal microvascular endothelial cells (hdMVEC)
Phosphorylation of eNOS on site serine 1176 (Ser1176) was assessed in hdMVEC ex vivo following incubation with diluted patient serum using the CytoFluor™ eNOS (Phospho-Ser1176) Fluorometric Cell-Based ELISA Kit (FLUO-CBP1542) from Assay Biotechnology (Fremont, CA, USA) according to the manufacturer’s instructions (Fig. 3a). Phosphorylation of eNOS on Ser1176 has been shown to result in increased NO production at basal levels, therefore maintaining microvascular function [14].
Briefly, cryopreserved pooled hdMVEC and the recommended cell culture media were purchased from PromoCell (Heidelberg, Germany). Cells of less than 6 passages were cultured in medium in T75 flasks until 80–90% confluence in a standard cell culture incubator (37 °C in a 5% CO2 humidified atmosphere). Cells were harvested using Accutase™ solution (Sigma-Aldrich, St. Louis, MO, USA) and seeded into 96-well plates coated with Attachment Factor (Thermo Fisher Scientific). After overnight cultivation, the supernatant was replaced by patient serum diluted (10%) in cell culture medium. Serum samples collected from different patients at the various time points (baseline, after 5 L of fluid administration, or postoperatively) were used for assaying. Cells were incubated for 4 h, then the supernatant was removed, and cells were fixed using 4% paraformaldehyde solution (Thermo Fisher Scientific). Subsequently, eNOS as well as phospho-eNOS activity were determined fluorometrically and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) according to the assay kit protocol.
Assessment of microvascular reactivity and nitric oxide (NO) bioavailability in vivo
Assessment of microvascular reactivity was performed similar to what was described by Kim et al. [6]. Following induction of anesthesia, a near-infrared spectroscopy (NIRS) sensor (Nonin Medical, Inc., Plymouth, MN, USA) was placed on the patient’s left forearm and connected to a Nonin 7600 4-Channel Regional Oximeter (Nonin Medical, Inc.) to measure muscle tissue oxygen saturation (StO2). Dynamic response of microvascular reactivity was assessed by an arterial vascular occlusion test (VOT) performed every 30 min. A blood pressure cuff was placed around the patient’s ipsilateral upper arm (Fig. 4a). For VOT, the cuff was inflated to 50 mmHg above the systolic arterial blood pressure. The pressure was maintained for 4 min, and afterwards, the cuff was rapidly and completely deflated. All data were initially stored in the internal memory of the device and transferred to an external computer for further analysis at the end of surgery. Data were visualized, and the following parameters were derived from the resulting VOT curve (Fig. 4b): baseline StO2 before VOT maneuver (PreVOT [%]), slope of tissue desaturation during VOT maneuver (DesatVOT [%/min]), time to recover to baseline StO2 following deflation of the cuff (RecovVOT [sec]), and size of reactive hyperemic area following VOT (HyperemicVOT [%*sec]).
Statistical and bioinformatical analysis
Data were transferred into MS Excel (Microsoft Corp., Redmond, CA, USA). Statistical analysis and visualization were performed using GraphPad PRISM 8 (La Jolla, CA, USA). All data are presented as median values with 25th and 75th percentile. Significance of differences between samples from two different time points was tested using the Wilcoxon signed-rank test. Changes over time in intraoperative assessments of microvascular reactivity were compared with the baseline using the Friedman test in case of complete data sets from all included patients. In case of missing values at various time points, Friedman test is inappropriate and Wilcoxon signed-rank test was used instead. Associations between different parameters were assessed using the Spearman rank correlation coefficient. P values < 0.05 were considered statistically significant.
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.