Section 1：Splanchnic microcirculatory changes during hemorrhagic shock and resuscitation in a rat model
1.1 Animal study I: Microcirculatory changes in multiple splanchnic organs during hemorrhagic shock and saline resuscitation.
Experimental animals
A total of 72 male Wistar rats were used (body weight 250 ± 50 g; Biolasco Taiwan Co., Taipei, Taiwan) in this experiment. The rats were raised on a 12-h light–dark cycle and had free access to water and food. Before conducting the experiments, all experimental procedures were approved by the Institutional Animal Care and Use Committee of National Taiwan University and performed in accordance with its guidelines.
Anesthesia and surgical procedure
The rats were anesthetized and prepared as described in our previous study (Yeh et al., 2012). A tracheostomy was performed, and then a 14G catheter (Surflo; Terumo Corporation, Laguna, Philippines) was inserted into the trachea to maintain
spontaneous breathing. The anesthesia was maintained using 1.2% isoflurane through the tracheal tube. Subcutaneous atropine 0.05 mg/kg in 10 mL/kg of saline was injected for replacement of water evaporization from surgical open wound and to prevent airway secretion. The body temperature was continuously monitored rectally, and a warmer pad was applied to maintain the rectal temperature between 36 and 37
°C. Polyethylene catheters (PE-50; Intramedic 7411, Clay Adams, Parsippany, NJ, USA) were inserted into the right common carotid artery and external jugular vein.
The right common carotid artery catheter was used to continuously monitor the MAP and heart rate (HR), and to withdraw blood to establish the state of hemorrhagic
13
shock. The external jugular vein catheter was used to infuse resuscitation fluid. A long midline laparotomy was performed to expose splanchnic organs including the liver, left kidney, and a segment of the terminal ileum (approximately 6 to 10 cm proximal to the ileocecal valve). A 2-cm section was incised on the antimesenteric aspect of the intestinal lumen by using a high-frequency desiccator (Aaron 900; Bovie Aaron Medical, St. Petersburg, FL, USA) to carefully expose the opposing mucosa for Examination of the microcirculatory blood flow. Nearby intestinal serosal muscular layer (at the midline of antimesenteric aspect) and the central Peyer's patch (identified by visualize the lymph nodes) were also identified for examination of the
microcirculatory blood flow. Moreover, the right gracilis musclewas exposed for measuring microcirculatory changes relative to those of the splanchnic organs (Fig 1).
The exposed viscera and tissue were kept moist hourly with saline (37 °C 0.5 mL of aerosolized 0.9% saline).
Hemorrhagic shock and fluid resuscitation protocol
After completion of the surgical procedures, the animals were allowed to stabilize for 30 min before the baseline hemodynamic measurements were performed (baseline condition was considered stable when all measurement values remained at 10% for 15 min; T0). After the baseline hemodynamic measurements were collected, the
concentration of inhaled isoflurane was decreased to 0.7% to prevent over anesthesia for hemorrhaging animal without further surgical stimulation, and the rats were randomly assigned to either a sham operation (S) group, hemorrhagic shock (H) or fluid resuscitation with 0.9% saline (R) group. In the H group, hemorrhagic shock was initiated through controlled blood withdrawal via the right carotid arterial cannula (3 times of 10 mL/kg per 5 min; total blood loss of 30 mL/kg during 15 min). Further macrocirculatory and microcirculatory monitoring were measured according to the
14
time points shown in Fig. 2. In the S group, the rats received the same surgical
preparation without undergoing blood withdrawal. The R group was resuscitated by a total of 30 mL/kg of 0.9% saline after hemorrhagic shock for 30 min.
Evaluation of splanchnic microcirculatory blood flow and oxygen saturation changes secondary to hemorrhagic shock
A full-field laser perfusion imager (MoorFLPI; Moor Instruments, Ltd., Devon, UK) was used from the baseline (T0) to continuously quantify microcirculatory blood flow intensity among multiple splanchnic organs. The imager uses LSCI, which exploits the random speckle pattern that is generated when tissue is illuminated by a laser light. The random speckle pattern changes when blood cells move within the region of interest (ROI). When the level of movement is high (high flow), the changing pattern becomes more blurred, and the contrast in that region decreases accordingly.
Therefore, low contrast represents high flow and high contrast represents low flow.
The contrast image is processed to produce a 16-color coded image that correlates with blood flow in the tissue (e.g., blue is defined as low flow and red as high flow).
The microcirculatory blood flow intensity of each ROI was recorded as a perfusion unit (PU), which is related to the product of the average speed and concentration of the red blood cells moving in the tissue sample volume (i.e., blood cell flux or perfusion). The images were recorded and analyzed in real time by using the MoorFLPI Version 3.0 software (Moor Instruments, Ltd.). Six separate ROIs were established on the liver, left kidney, mucosa, serosal muscular layer, Peyer's patch, and right gracilis muscle. Details of selecting and setting ROIs were as following:
1. The left liver is identified by the hepatic fissure and falciform ligament. The hepatic ROI was set 1.5 cm left to the hepatic fissure.
2. The left kidney is easily identified by direct visualization.
15
3. The selected terminal ileum is about 6 to 10 cm proximal to the ileocecal valve in each animal. The length of section was fixed in each animal (2-cm; performed on the antimesenteric aspect of the intestinal lumen). In addition, the choice of specific interested region of intestinal tissue was the same for each of the rats as the below: First, the ROI of mucosa was set to cover as much of the mucosa as possible. The advantage of using a high-frequency desiccator to expose the intestine is that it is able to confine the surgical trauma to small and restricted area. Accordingly, the ROI of the mucosa was carefully selected not to cover the outmost margin. The choice of intestinal serosal muscular layer was at the midline of antimesenteric aspect (one to 2 cm away from the mucosa). The Peyer’s patch was identified by visualizing the clusters of lymph nodes.
4. This multiple organ model is involved with some stretching forces to fully expose the terminal ileum. In addition, the mesenteric perfusion has a gray zone that the distal portion of supplied tissue is more susceptible to stretch-induced decrease of blood flow. Therefore, the region of interests were selected in the central portion of exposed tissue and we were carefully to avoid covering the ROIs over the outmost margin of each tissue.
5. The skin (2 cm incision) to expose the gracilis muscle was about 3 cm lateral to the midline of abdomen (the midline could be identified by the xiphoid bone and anus).
6. The ROI was set up as a square with the length approximately 1.0 to 1.5 cm to cover as much of the exposed tissues as possible.
The tissue oxygen saturation was measured using a white light spectroscopy needle probe (moorVMS-LDF2; Moor Instruments, Ltd.) with a white LED for illumination, which emitted light with wavelengths between 450 and 700 nm (Liu et al., 2011). To minimize sampling bias, tissue oxygen saturation was measured and
16
recorded as an average of 5 points in each exposed target organ. The microcirculation, including blood flow intensities and tissue oxygen saturation between the 2 groups, were compared at the following time points: 0 min (baseline; T0), 75/105 min (1 h after shock or resuscitation; T1), 135/165 min (2 h after shock or resuscitation; T2), and 195/225 min (3 h after shock or resuscitation; T3) (Fig. 2). Microcirculatory blood flow intensity was recorded as an arbitrary PU, and the tissue oxygen saturation was recorded as a percentage (%). Because different organs may have different baseline states of both macrocirculatory and microcirculatory blood flow intensity and tissue oxygen saturation, the percent changes of the MAP, microcirculatory blood flow intensity, and tissue oxygen saturation at T1, T2, and T3 were compared to the T0
baseline values.
The total 72 rats in the S, H and R groups were equally assigned to the following 3 subgroups: (1) measurements of changes in microcirculatory blood flow intensity;
(2) measurements of tissue oxygen saturation in the intestinal mucosa, serosal muscular layer, Peyer's patch, and right gracilis muscle; and (3) measurements of tissue oxygen saturation in the liver and kidney.
17
1.2 Animal study II: Effects of different types of fluid resuscitation for
hemorrhagic shock on splanchnic organ microcirculation and renal reactive oxygen species formation.
Experimental animals
A total of 96 male Wistar rats (body weight = 250 ± 30 g; Biolasco Taiwan Co., Taipei, Taiwan) were used in the second animal experiment. The rats were raised on a 12-h light–dark cycle and had a free access to water and food. All experimental procedures were approved by the Institutional Animal Care and Use Committee of National Taiwan University and were conducted in accordance with its guidelines.
Part I - changes of microcirculatory blood flow intensity in splanchnic organs Anesthesia and surgical procedure
The anesthesia and surgical protocol were the same as those in the above mentioned animal study I. Accordingly, a tracheostomy was performed, and then a 14G catheter (Surflo; Terumo Corporation, Laguna, Philippines) was inserted into the trachea to maintain spontaneous breathing. Anesthesia was maintained using 1.2 % inhaled isoflurane through the tracheal tube. Subcutaneous atropine (0.05 mg/kg in 10 mL/kg of saline) was injected to limit the rate of moisture evaporation from the surgical open wound and to prevent airway secretion. The body temperature of the rats was
continuously monitored rectally, and a warmer pad was applied to maintain it between 36 and 37 °C. Polyethylene catheters (PE-50; Intramedic 7411, Clay Adams,
Parsippany, NJ, USA) were inserted into the right common carotid artery and external jugular vein. The catheter in the right common carotid artery was used to continuously monitor the MAP and HR, and to withdraw blood for inducing hemorrhagic shock.
The external jugular vein catheter was used to infuse resuscitation fluid.
A midline laparotomy was performed to exteriorize splanchnic organs, including
18
the liver, left kidney, and a segment of the terminal ileum (approximately 6–10 cm in length, proximal to the ileocecal valve). A 2-cm section was made on the
antimesenteric aspect of the intestinal lumen by using a high-frequency desiccator (Aaron 900; Bovie Aaron Medical, St. Petersburg, FL, USA) for carefully exposing the opposing mucosa to study microcirculation. Furthermore, the nearby intestinal serosal muscular layer (at the midline of the antimesenteric aspect) and the central Peyer’s patch (identified by visualizing the lymph nodes) were identified to study microcirculation. Moreover, the right gracilis muscle was exposed for monitoring microcirculatory changes relative to those in the splanchnic organs. The exposed viscera and tissue were kept moist by treating them every hour with 0.5 mL of prewarmed to 37 °C aerosolized 0.9% saline.
Evaluation of microcirculatory blood flow
The microcirculatory blood flow intensity was assessed using the LSCI technique and the setup was similar to that employed in the animal study I. A full-field laser
perfusion imager (MoorFLPI; Moor Instruments, Ltd., Devon, UK) was used to continuously record the intensity of microcirculatory blood flow in the splanchnic organs, starting from the baseline. The imager uses LSCI, in which a random speckle pattern is generated when a tissue is illuminated by a laser light. The random speckle pattern changes when blood cells move within the ROIs. When the level of movement is high (high flow), the changing pattern becomes more blurred, and the contrast in that particular region declines accordingly. Therefore, low contrast is associated with high flow, and high contrast is associated with low flow. The contrast image is
processed to produce a 16-color-coded image that is associated with the blood flow in the tissue (e.g., blue indicates low flow and red, high flow). The microcirculatory blood flow intensity of each ROI was recorded as a PU, which is equal to the product of the average speed and concentration of red blood cells moving in the tissue sample
19
volume (i.e., blood cell flux or perfusion). The images were recorded and analyzed in real time using the MoorFLPI Version 3.0 software (Moor Instruments, Ltd.). Six individual ROIs were defined on the liver, left kidney, intestinal mucosa, serosal muscular layer, Peyer’s patch, and right gracilis muscle (identical to the animal study I). Microcirculatory blood flow intensity was recorded as an arbitrary PU.
Hemorrhagic shock and fluid resuscitation protocols
A total 60 rats were randomly assigned to the following six groups (n = 10 in each group): (1) the sham group, in which the rats received all surgical procedures except blood withdrawal and fluid resuscitation; (2) the control group, in which the rats underwent hemorrhaging but no fluid resuscitation; (3) the NS group, in which the rats were resuscitated with 0.9 % saline; (4) the HTS group, in which the rats were resuscitated with 3 % HTS; (5) the GEL group, in which the rats were resuscitated with 4 % succinylated GEL (Gelofusine®, B. Braun, Taiwan); and (6) the HES group, in which the rats were resuscitated with 6 % HES 130/0.4 (Voluven®, Fresenius-Kabi, Bad Homberg, Germany).
The timeline of part I of this experiment is shown in Figure 3a. After completion of surgery, the rats were allowed to hemodynamically stabilize for 30 minutes before the baseline hemodynamic measurements were recorded (baseline condition was considered stable when all measurement values remained at 10 % for 15 minutes;
defined as 0’ minutes; T0). At T0, the concentration of isoflurane was lowered to 0.7 % to prevent overanesthesia in the rats without further surgical stimulation, and
hemorrhagic shock was then induced through controlled blood withdrawal via a right carotid arterial cannula with total blood loss of 30 mL/kg from 15’ to 60’ (T1)
minutes. Fluid resuscitation was administrated from 60’ to 90’ minutes, and the volume of fluid resuscitation was identical to that of blood withdrawn. Two hours
20
after fluid resuscitation, part I of the experiment was considered complete (T2; at 210’
minutes in the rats receiving fluid resuscitation and at 180’ min in the rats in the sham and control groups; Fig. 3a). Hemodynamic measurements of both macrocirculatory and microcirculatory variables were compared among the groups at T0, T1, and T2
(Fig. 3a). Because different organs may have different baseline states of both macrocirculatory and microcirculatory conditions, the percent changes in the
microcirculatory blood flow intensity at T1 and T2 were compared with the T0 baseline values.
The first 0.2 mL of blood withdrawn at 15’ minutes and the final 0.2 mL of blood withdrawn at T1 were analyzed using arterial blood gas analysis as the baseline status and shock status, respectively. At the end of the measurement (T2), arterial blood gas analysis was repeated to evaluate the effects of fluid resuscitation.
Part II. In vivo renal reactive oxygen species formation
For measuring in vivo kidney ROS activity during hemorrhagic shock and fluid resuscitation, 36 rats underwent the same hemorrhagic shock and fluid resuscitation (N = 6, in each group) protocols as did the rats used for investigating microcirculatory changes in the splanchnic organs with two modifications. First, the rats were
anesthetized with a subcutaneous urethane injection (1.2 g/kg) instead of volatile anesthesia because in vivo measurement was conducted in a closed box that did not allow the entry of a volatile anesthesia breathing circuit. In addition, volatile
anesthesia may suppress ROS formation because of its strong antioxidant properties
21
(De Wolf and Hendrickx, 2013). Second, pilot experiments showed that rats receiving anesthesia with subcutaneous urethane could survive only if the volume of blood loss was lowered. Therefore, hemorrhagic shock was induced by withdrawing a lesser amount of blood (20 mL/kg; Fig. 3b).
The method for detecting chemiluminescence (CL) from the organ surface after intrarenal arterial infusion of a superoxide anion probe,
2-methyl-6-[4-methoxyphenyl]-3,7-dihydroimidazo-[1,2-a]-pyrazin-3-one hydrochloride (MCLA) (TCI-Ace; Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan) (Chien et al., 2001, Yang et al., 2014), was adapted to demonstrate ROS production in kidneys subjected to ischemia-reperfusion injury. For excluding photon emission from sources other than the kidney, the rats were housed in a dark box with a shielded plate. Only the renal window was left unshielded and was positioned under a reflector, which reflected the photons from the exposed kidney surface onto the detector area. A single dose of N,N9-dimethyldiacridium (1 mM in 0.1 mL) (lucigenin; Sigma) or a continuous infusion of MCLA (0.2 mg/mL per h) was administered through an intrarenal arterial catheter. The lucigenin- or MCLA-enhanced CL signal from the renal surface was measured continuously during administration by using a CL analyzer (CLD-110, Tohoku Electronic Industrial Co., Sendai, Japan). The ROS response was directly evaluated from the renal surface through intrarenal arterial infusion of MCLA (0.2
22
mg/mL/h) (Chien et al., 2001). At T2, the changes in ROS formations from baseline were compared.
23
Section 2：Predicting stroke volume and arterial pressure fluid responsiveness in liver cirrhosis patients by using dynamic preload variables
Patients
Before conducting this clinical study, this study was approved by the Research Ethics Committee of National Taiwan University Hospital, Taipei, Taiwan, (Chairperson:
Professor Hong-Nerng Ho) on 4 October 2013 (201309024RIND). After obtaining written informed consent from all patients, adult recipients of a living donor
orthotopic liver transplant were consecutively enrolled from November 2013 to April 2015. The exclusion criteria were a history of pulmonary resection, chronic
respiratory insufficiency, left ventricular ejection fraction (LVEF) of <60% or pulmonary hypertension, and arrhythmia (NCT01971333).
Anesthesia
General anesthesia induction is performed by using intravenous fentanyl (1.2 µg kg−1), etomidate (0.3 mg kg−1), and cisatracurium (0.15 mg kg−1). Anesthesia was then maintained in a standard manner with desflurane in an air/oxygen mixture and
intravenous infusions of fentanyl and cisatracurium. During surgery, the anesthetic depth was maintained by keeping the bispectral index (BIS) between 40 and 60. For maintenance of normocapnia, mechanical ventilation was set to a tidal volume of 8 mL kg−1, a respiratory rate of 10–15 min−1, and a positive end-expiratory pressure of 5
24
cmH2O. The respiratory settings were not changed throughout the measurements.
Hemodynamic assessment
Each patient received standard monitoring by using a Philips Intellivue MP 70 monitor (Philips, Suresnes, France). After general anesthesia, a triple-lumen 5.5-Fr catheter (Arrow central venous catheter; Teleflex Life Sciences Ltd., Athlone, Ireland) was placed into the right internal jugular vein. A 4-Fr thermistor-tipped arterial
catheter (Pulsiocath thermodilution catheter; Pulsion Medical Systems, Munich, Germany) was inserted into the right femoral artery, advanced to the abdominal aorta, and connected to the PiCCOplus™ system monitor (Version 7.0; Pulsion Medical Systems). The PiCCO system is using the transpulmonary thermodilution technique to generate the cardiac index, SV index (SVI; SV divided by the body surface area for normalisation), and SVRI, which are comparable to those generated using the
pulmonary artery catheter (Della Rocca et al., 2002a, Hofer et al., 2005, Della Rocca et al., 2002b).Continuous SVI measurement was initiated after the system was initially calibrated by injecting 20 mL of ice-cold normal saline (< 8°C ) into the central venous catheter (transpulmonary thermodilution) three times, and further calibration was performed hourly. The cardiac index was calculated as follows: SVI × heart rate.
The Masimo Radical 7 co-oximeter (version 7.8; Masimo Corp., Irvine, CA, USA)
25
uses a finger clip to measure arterial oxygen saturation concentration noninvasively on the basis of transcutaneous multiwavelength analysis. The Masimo sensor was attached to the right index finger according to the manufacturer’s instructions and was on the side opposite from the intravenous catheter.
Dynamic preload variables
SVV and PPV were calculated according to the following formula: SVV (%) = [(SVmax − SVmin)/SVmean] × 100 and PPV (%) = [(PPmax − PPmin)/PPmean] × 100. Using the PiCCOplus™ system, we measured SVmax and SVmin (or PPmax and PPmin) as the mean values of the four extreme values of SV (or PP) during measurement from the femoral artery for 30 s. The SVmean (or PPmean) was recorded as the average value during this period.
The PVI, which is based on the perfusion index, was obtained from the Masimo Radical 7 SET co-oximetery. The PVI was calculated as [(perfusion indexmax − perfusion indexmin)/ perfusion indexmax] × 100 over a period sufficient to include multiple respiratory cycles.
Vascular tone variables
Vascular tone variables including the SVRI, perfusion index, and PPV/SVV ratio were measured. The SVRI was calculated as (MAP – CVP) × 80/cardiac index. The perfusion index was calculated as the ratio of pulsatile to non-pulsatile blood volumes
26
and was automatically calculated using the Masimo Radical 7 SET co-oximetery.
Fluid challenge criteria, timing, and the definition of stroke volume and arterial pressure fluid responders
After the PiCCO monitor was set up, the patients were allowed to stabilize for 15 min.
Subsequently, they received a fluid challenge of 10 mL kg−1 0.9% saline within 15 min during the surgical preparation before skin incision. An additional saline challenge was administered 2 h after the surgical incision. During the dissection phase, each patient received basal 0.9% saline infusion at a rate of 5 mL kg−1 h−1. Hemodynamic data, namely the HR, MAP, CVP, SVI, PPV, SVV, PPV/SVV ratio,
Subsequently, they received a fluid challenge of 10 mL kg−1 0.9% saline within 15 min during the surgical preparation before skin incision. An additional saline challenge was administered 2 h after the surgical incision. During the dissection phase, each patient received basal 0.9% saline infusion at a rate of 5 mL kg−1 h−1. Hemodynamic data, namely the HR, MAP, CVP, SVI, PPV, SVV, PPV/SVV ratio,