ORTHOGONAL POLARIZATION SPECTROSCOPY TO DETECT MESENTERIC HYPOPERFUSION—Electronic Supplement

ICM-2007-00173.R2

Anesthesia and monitoring

Anesthesia and monitoring have been described in detail elsewhere [8]. Briefly, 13 domestic pigs (weight 30 [23-39] kg) were anesthetized, intubated and ventilated with 70% air and 30% oxygen. Anesthesia and muscle paralysis were maintained with continuous infusion of midazolam 0.5 mg/kg/hr, fentanyl 20 mcg/kg/hr, and pancuronium 0.5 mg/kg/hr. Intraoperative blood loss was replaced with colloids. After surgery, the rate of the crystalloid infusion was set to 20 mL/kg/h. Central venous, carotid and pulmonary artery pressures and pulmonary artery occlusion pressure were recorded and displayed continuously on a multi-modular monitor (Datex-Ohmeda S/5, Datex-Ohmeda Division, Helsinki, Finland). Heart rate was continuously measured from the ECG. Cardiac output was measured using the thermodilution method. Central body temperature was measured using the thermistor of the pulmonary artery catheter, with the aim to keep the core temperature between 38°C and 39°C. The adequacy of the fluid regimen was assessed clinically by using central hemodynamics and diuresis.

Quantification of perfusion

To quantify the perfusion, capillary density was assessed by counting vessels crossing four equidistant vertical and horizontal lines, e.gi.e.,. a region of interest (ROI). A ROI consisted of 9 squares or 8 lines that could be crossed by capillaries. Capillary density was then defined as the total number of vessels crossing the equidistant lines of the ROI. Furthermore, the total number of all mucosal villi and the number of perfused mucosal villi was counted and the flow pattern was chosen from three categories: “High flow” for a constant, high flow in most of the analyzed capillaries; “intermediate/stagnant flow” for a predominantly moderate flow impairment, and “no flow” for a typical picture of “stop flow” in the analyzed capillaries. In a last step, the homogeneity of the image was evaluated. For this, the image was scored and then classified according to the flow pattern; e.g., if all capillaries showed the same flow pattern, the category was “homogeneous”; if there were areas with high flow adjacent to areas with intermediate/stagnant flow or no flow, the category was “heterogeneous.”

After having agreed on the method, both investigators first quantified five sequences, compared the results, and agreed again on the method where differences were found. Afterwards all sequences were analyzed individually by both investigators (n=268). Next, the two investigators re-analyzed 12 randomly selected sequences each, this time using the very same picture frames (but not necessarily the same ROI) that had been used by the other investigator during his first analysis. Finally, the same 24 picture frames were analyzed once more, but this time both investigators used the same region of interest. This approach was used to control for a possible influence of the selected sequences and/or the selected region of interest.

To test the relevance of measurement site for OPS, further OPS recordings were performed in sublingual location in eight healthy volunteers, who all gave written informed consent. The sublingual recordings were blindly analyzed by the same two investigators.

Further statistical remarks

To assess the similarity of the two groups (blood flow reduction and controls) and between the analysis of the two investigators (OPS) at baseline, a Mann-Whitney U test (groups) and a Wilcoxon matched-pairs test (investigators) were used. Afterward, changes over time were analyzed separately in both groups, for the two investigators (OPS), and also at the three measurement sites (LDF) by a Friedman test. If significant, a Wilcoxon matched-pairs test and a Mann-Whitney test for paired differences (baseline minus flow reduction level), respectively, were used to detect the first time point when there was a significant difference compared with baseline. The same approach was used for differences between groups and investigators. Since there were three LDF measurement sites in mucosa and serosa, Mann-Whitney tests between sites were only performed when a previous Kruskal Wallis test was significant. To determine the significant differences in blood flow reduction levels, and between investigators and measurement sites, multiple comparisons were performed. Nevertheless, the commonly used significance level of p < 0.05 was applied because a certain level was only considered significant when it was fortified by demonstrating an increasing divergence, both as compared with baseline and between the groups below that level. If either divergence was not increasing, the difference was considered not to be significant.


Table ES1: Systemic hemodynamics and SMA blood flow

Reduction 0% 15% 30% 45% 60% 75% 90%

Time 0 min 45 min 90 min 135 min 180 min 225 min 270 min

Group R (n=8)

HR (bpm)# 117 (95-199) 120 (91-154) 117 (80-160) 124 (116-153) 145 (100-177) 144 (108-190) 159 (104-177)

MAP (mmHg)# 95 (61-101) 85 (68-109) 81 (75-110) 81 (68-108) 84 (73-97) 89 (70-100) 96 (93-133)

CI (ml/kg/min) 175 (151-239) 158 (122-217) 189 (122-217) 192 (122-243) 208 (116-263) 211 (122-296) 190 (134-289)

MPAP (mmHg) 19 (16-22) 19 (15-20) 19 (15-20) 20 (15-21) 23 (16-24) 23 (15-31) 24 (15-26)

CVP (mmHg) 5.0 (1.0-6.0) 4.5 (2.0-6.0) 4.5 (3.0-7.0) 4.5 (3.0-6.0) 5.0 (4.0-7.0) 5.0 (4.0-7.0) 5.0 (4.0-6.0)

PCWP (mmHg) 5.0 (3.0-6.0) 4.0 (4.0-7.0) 4.5 (4.0-7.0) 5.0 (3.0-7.0) 4.5 (4.0-5.0) 4.0 (3.0-7.0) 4,0 (2,0-7,0)

QSMA (ml/min/kg) 24 (18-31) 21 (17-34) 17 (13-23) 14 (11-19) 10 (9-14) 6 (5-11) 3 (2-5)

Group C (n=5)

HR (bpm)# 107 (81-153) 112 (91-136) 133 (101-153) 146 (103-181) 133 (97-164) 144 (118-188) 151 (139-198)

MAP (mmHg) 95 (69-101) 83 (66-100) 96 (66-121) 84 (70-126) 91 (69-95) 87 (64-104) 77 (62-105)

CI (ml/kg/min)# 165 (147-207) 166 (128-177) 185 (147-223) 217 (144-231) 217 (141-227) 221 (153-254) 233 (166-262)

MPAP (mmHg) 18 (14-33) 18 (15-27) 18 (15-24) 19 (17-24) 19 (17-21) 19 (17-22) 21 (16-22)

CVP (mmHg) 4.0 (3.0-8.0) 4.0 (3.0-8.0) 4.0 (3.0-7.0) 4.0 (4.0-7.0) 4.0 (4.0-7.0) 4.0 (4.0-7.0) 5.0 (4.0-7.0)

PCWP (mmHg) 4.5 (3.0-6.0) 4.0 (4.0-6.0) 5.0 (3.0-6.0) 5.0 (3.0-7.0) 5.0 (3.0-7.0) 5.0 (3.0-7.0) 5,5 (3,0-6,0)

QSMA (ml/min/kg) 25 (15-33) 23 (16-37) 22 (16-38) 22 (14-39) 23 (13-39) 23 (15-44) 24 (14-42)

Systemic hemodynamics. In group R, SMA flow was reduced in six steps of 15% using an occluder. In group C, the occluder was placed around the SMA but not inflated. HR: heart rate; MAP: mean arterial blood pressure; CI: cardiac index; MPAP: mean pulmonal arterial pressure; CVP: central venous pressure, PCWP pulmonary capillary wedge pressure, QSMA blood flow in the superior mesenteric artery. Results are presented as median (range). #p<0.05 using Friedman Test.

1 of 10

Figure Legends

Figure ES 1

Figures ES 1 a-d. Laser Doppler flow (LDF) of the three different laser Doppler probes on jejunal mucosa (Figures a + b) and jejunal serosa (Figures c + d). Group R is stepwise blood flow reduction, group C is control animals. Data are shown as median (range). # Wilcoxon test, compared to baseline, first p<0.05.

Figure ES 2

Figure ES 2a-d. Flow pattern as estimated by the two observers in the ileal mucosa in controls (Group C) and during stepwise reduction of superior mesenteric artery (SMA) blood flow (Group R). Data are shown as percent of analyzed sequences at baseline and after 15%, 30%, 45%, 60%, 75% and 90% reduction of SMA blood flow. § Mann-Whitney rank sum test vs. observer 2, p<0.05, for the different flow types, # Wilcoxon signed rank test vs. baseline, first p< 0.05 for the different flow types.

Figure ES 3

Figure ES 3a-d. Flow picture as estimated by the two observers in the ileal mucosa in controls (Group C) and during stepwise reduction of superior mesenteric artery (SMA) blood flow (Group R). Data are shown as percent of analyzed sequences at baseline and after 15%, 30%, 45%, 60%, 75% and 90% reduction of SMA blood flow.

Figure ES 4

Representative picture of two equal sequences for the two observers, taken from the post-analysis (a+b), and of two equal sequences of the sublingual analysis (c+d) . Note the different regions of interest chosen, as well as the different assessment of the numbers of vessels, villi, and perfused villi in the mucosal OPS compared to the better agreement in sublingual OPS.

1 of 10