The use of pulse pressure variation for predicting impairment of microcirculatory blood flow

Dynamic parameters of preload have been widely recommended to guide fluid therapy based on the principle of fluid responsiveness and with regard to cardiac output. An equally important aspect is however to also avoid volume-overload. This accounts particularly when capillary leakage is present and volume-overload will promote impairment of microcirculatory blood flow. The aim of this study was to evaluate, whether an impairment of intestinal microcirculation caused by volume-load potentially can be predicted using pulse pressure variation in an experimental model of ischemia/reperfusion injury. The study was designed as a prospective explorative large animal pilot study. The study was performed in 8 anesthetized domestic pigs (German landrace). Ischemia/reperfusion was induced during aortic surgery. 6 h after ischemia/reperfusion-injury measurements were performed during 4 consecutive volume-loading-steps, each consisting of 6 ml kg−1 bodyweight−1. Mean microcirculatory blood flow (mean Flux) of the ileum was measured using direct laser-speckle-contrast-imaging. Receiver operating characteristic analysis was performed to determine the ability of pulse pressure variation to predict a decrease in microcirculation. A reduction of ≥ 10% mean Flux was considered a relevant decrease. After ischemia–reperfusion, volume-loading-steps led to a significant increase of cardiac output as well as mean arterial pressure, while pulse pressure variation and mean Flux were significantly reduced (Pairwise comparison ischemia/reperfusion-injury vs. volume loading step no. 4): cardiac output (l min−1) 1.68 (1.02–2.35) versus 2.84 (2.15–3.53), p = 0.002, mean arterial pressure (mmHg) 29.89 (21.65–38.12) versus 52.34 (43.55–61.14), p < 0.001, pulse pressure variation (%) 24.84 (17.45–32.22) versus 9.59 (1.68–17.49), p = 0.004, mean Flux (p.u.) 414.95 (295.18–534.72) versus 327.21 (206.95–447.48), p = 0.006. Receiver operating characteristic analysis revealed an area under the curve of 0.88 (CI 95% 0.73–1.00; p value < 0.001) for pulse pressure variation for predicting a decrease of microcirculatory blood flow. The results of our study show that pulse pressure variation does have the potential to predict decreases of intestinal microcirculatory blood flow due to volume-load after ischemia/reperfusion-injury. This should encourage further translational research and might help to prevent microcirculatory impairment due to excessive fluid resuscitation and to guide fluid therapy in the future.


INTRODUCTION
Background 3 a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale. b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the stud s relevance to human biology.
Objectives 4 Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.

Ethical statement 5
Indicate the nature of the ethical review permissions, relevant licences (e.g. Animal [Scientific Procedures] Act 1986), and national or institutional guidelines for the care and use of animals, that cover the research.

Study design 6
For each experiment, give brief details of the study design including: a. The number of experimental and control groups. b. Any steps taken to minimise the effects of subjective bias when allocating animals to treatment (e.g. randomisation procedure) and when assessing results (e.g. if done, describe who was blinded and when). c. The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out.

7
For each experiment and each experimental group, including controls, provide precise details of all procedures carried out. For example: a. How (e.g. drug formulation and dose, site and route of administration, anaesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Provide details of any specialist equipment used, including supplier(s). b. When (e.g. time of day). c. Where (e.g. home cage, laboratory, water maze). d. Why (e.g. rationale for choice of specific anaesthetic, route of administration, drug dose used).
Experimental animals 8 a. Provide details of the animals used, including species, strain, sex, developmental stage (e.g. mean or median age plus age range) and weight (e.g. mean or median weight plus weight range). b. Provide further relevant information such as the source of animals, international strain nomenclature, genetic modification status (e.g. knock-out or transgenic), genotype, health/immune status, drug or test naïve, previous procedures, etc.
Housing and husbandry 9 Provide details of: a. Housing (type of facility e.g. specific pathogen free [SPF]; type of cage or housing; bedding material; number of cage companions; tank shape and material etc. for fish). b. Husbandry conditions (e.g. breeding programme, light/dark cycle, temperature, quality of water etc for fish, type of food, access to food and water, environmental enrichment). c. Welfare-related assessments and interventions that were carried out prior to, during, or after the experiment.
Sample size 10 a. Specify the total number of animals used in each experiment, and the number of animals in each experimental group. b. Explain how the number of animals was arrived at. Provide details of any sample size calculation used. c. Indicate the number of independent replications of each experiment, if relevant.
Allocating animals to experimental groups 11 a. Give full details of how animals were allocated to experimental groups, including randomisation or matching if done. b. Describe the order in which the animals in the different experimental groups were treated and assessed.

12
Clearly define the primary and secondary experimental outcomes assessed (e.g. cell death, molecular markers, behavioural changes).

13
a. Provide details of the statistical methods used for each analysis. b. Specify the unit of analysis for each dataset (e.g. single animal, group of animals, single neuron). c. Describe any methods used to assess whether the data met the assumptions of the statistical approach.

Baseline data 14
For each experimental group, report relevant characteristics and health status of animals (e.g. weight, microbiological status, and drug or test naïve) prior to treatment or testing. (This information can often be tabulated).
Numbers analysed 15 a. Report the number of animals in each group included in each analysis. Report absolute numbers (e.g. 10/20, not 50% 2 ).
b. If any animals or data were not included in the analysis, explain why.

Outcomes and estimation
16 Report the results for each analysis carried out, with a measure of precision (e.g. standard error or confidence interval).
Adverse events 17 a. Give details of all important adverse events in each experimental group. b. Describe any modifications to the experimental protocols made to reduce adverse events.

Additional file 2: Additional comments on ARRIVE Guidelines
This document contains additional comments on ARRIVE Guidelines.
Additional comments on ARRIVE Guidelines: 1. The abstract includes a summary of background, objectives and gives specific information to the species and methods used as requested by the ARRIVE Guidelines.
2. In the introduction we have included an overview of the scientific background and previous work relevant to this subject to provide adequate information to explain the purpose of this study. We have included statements on the usefulness of our experimental model and clearly stated the aim of this study as requested by the ARRIVE Guidelines.
3. In the declaration we have included information on ethical approval including reference-no. and

Additional file 3: Details of surgical procedures
This file contains details of surgical procedures.

Details of surgical procedures:
Surgical preparation was carried out according to standardized preparation techniques. The carotid artery was exposed and an 8 Fr. introducer sheath was inserted for placement of a Millar catheter in the ascending aorta. The femoral artery was exposed and an 8 Fr. introducer sheath was inserted for placement of a Millar catheter in the femoral artery. A 7.5 Fr. venous catheter and 8 Fr. introducer sheath were inserted in the right sided internal jugular vein for volume and drug application. A median laparotomy was performed. The descending aorta was exposed for graft implantation and a flow-probe was placed around the descending aorta proximal of the graft landing zone. The peritoneum was fenestrated in the area of the terminal ileum.
A 15-20 cm segment of the terminal ileum was defined for measurement of intestinal microcirculatory blood flow. The chosen ileum segment was marked with loosely attached vessel loops for identification whereat meticulous caution was taken not to affect perfusion due to tension or compression of the segment. Between baseline measurement and the measurement 6 h after ischemia/reperfusion, the ileum segment was left intraperitoneal. For Laser-Speckle-Contrast-Imaging measurements the ileum segment was carefully led on a humidified compress outside the peritoneum. 10 minutes were allowed for stabilization after extraperitoneal placement of the segment.
Meticulous caution was paid not to distort mesenteric vessels and before each measurement the segment was carefully checked for perfusion defects. Size of peritoneal fenestration was chosen wide enough not to impair perfusion and eventually enlarged if necessary. Between the volume loading steps after ischemia/reperfusion, the ileum segment was covered with humidified and warmed compresses to prevent heat and moisture loss. Undisturbed perfusion and lack of any compression or tension was carefully checked prior each measurement.
Aortic hybrid-graft implantation was performed using a hybrid-graft device combining a proximal stent graft and a distal multi-branched graft for re-implantation of the coeliac trunk (TC), the superior mesenteric artery (SMA) and both renal arteries (LRA, RRA).
The infra-diaphragmatic aorta was exposed through retroperitoneal access. After infracoeliac cross-clamping, the TC was divided and the proximal stent-grafted part of the graft was introduced into the descending aorta via the former coeliac ostium and carefully extracted. The coeliac, superior mesenteric, and renal arteries were successively connected to the corresponding side branches of the graft. Finally, iliac arteries (right iliac artery (RIA) and left iliac artery (LIA)) were anastomosed. Times of ischemia for TC, SMA, RRA, LRA, RIA, LIA are given as Additional File (Additional Table 1).

Additional file 4: Additional Tables
This file contains information on vessel ischemia duration (Additional Table 1) as well as statistical details (Additional Table 2a-h). In addition, pairwise comparison of macroand microcirculation with inclusion of baseline values are given in Additional Table 3.
Additional Table 1 Duration of vessel ischemia was not intended as standardized ischemia times but was dependent on the surgical techniques used. Additional Table 1 presents mean ischemic times for the different vessels.
Additional Additional Table 2a-h   Additional Table 2 a-h contain detailed information on general linear mixed model analyses for mean Flux (2a), cardiac output (2b), stroke volume (2c), heart rate (2d), mean arterial pressure (2e), central pulse pressure variation (2f) as well as on receiver operating characteristic analysis using central pulse pressure variation (2g) as well as peripheral pulse pressure variation (2h).
Additional       Additional Table 3 Pairwise comparison of macro-and microcirculation with inclusion of baseline values of mean microcirculatory blood flow (mFlux), cardiac output (CO), stroke volume (SV), heart rate (HR), mean arterial pressure (MAP) and central pulse-pressure-variation (PPV). Data are presented as estimated marginal means with 95 % confidence intervals. Pairwise p-values compared to previous measurement step are given. Points of measurements are baseline conditions prior induction of ischemia/reperfusion (baseline), 6 h after ischemia/reperfusion (I/R) and volume loading steps 1-4 6 h after ischemia/reperfusion (VLS1-4). Additional