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.
In this study, we established a rat model that could simultaneously reveal
microcirculatory changes among multiple organs during hemorrhagic shock. We found that (1) hemorrhagic shock induced a heterogeneous reduction in
microcirculatory blood flow intensity among splanchnic organs, with the intestinal mucosa being the most vulnerable; (2) during the acute phase of hemorrhagic shock, fluid resuscitation restored the MAP and improved the microcirculatory blood flow intensity impairment of the kidney, but the intestinal microcirculatory blood flow intensity remained compromised, especially in the intestinal mucosa; (3) hemorrhagic shock induced a greater homogeneous reduction of tissue oxygen saturation values among the various splanchnic organs than that of gracilis muscle.
The major difference between this study and previous studies investigating
microcirculatory changes after hemorrhagic shock is the usage of LSCI. The LSCI technique enables full-field scanning in near real time; therefore, it enables
simultaneous measurement of changes in microcirculatory blood flow intensity among multiple organs (van Iterson et al., 2012, Sordia et al., 2004, Morini et al.,
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2000, Beilman et al., 1999). Most previous studies have focused on microcirculation in only one or 2 specific organs and have not sufficiently investigated the
microcirculatory changes in multiple organs secondary to hemorrhagic shock.
Moreover, we used LSCI to investigate microcirculation for 2 additional reasons.
First, by using LSCI, a larger ROI can be set, avoiding the inaccurate results that can occur when choosing an inappropriately small region for monitoring. Previous studies have reported that LSCI reduces intersite and interindividual variability and may provide an improved reproducibility of microcirculation comparing to other
techniques such as laser Doppler flowmetry or sidestream dark-field (SDF) imaging (Tew et al., 2011, Roustit et al., 2010, Sturesson et al., 2013, Rousseau et al., 2011).
Second, the microcirculatory blood flow of viscera is likely obstructed by the physical attachment of the probes used in other techniques. LSCI is noninvasive, eliminating artifact contact (Draijer et al., 2009).
This study showed that the microcirculatory blood flow intensities of the liver, kidney, and gracilis muscle were the least affected by hemorrhaging and that the intestine, especially the mucosa, was the most vulnerable organ. Recently, Vajda at el.
found that the intestine was more sensitive to hemorrhaging than the heart in a swine model of hemorrhagic shock (Vajda et al., 2004). Additionally, we found that, with heterogeneous impairment of microcirculatory blood flow intensity within the
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intestine, the mucosa was more susceptible to hemorrhaging than the serosal muscular layer and Peyer’s patch. Similarly, Dubin et al. used SDF imaging in a sheep model of hemorrhagic shock and found that greater decreases in capillary density and increases in heterogeneity, implying a higher susceptibility to hemorrhaging, occurred in the ileal mucosa than in the ileal serosa and sublingual mucosa (Dubin et al., 2009).
However, other splanchnic organs were not compared in that report.
Previously, Sand et al. also used LSCI to assess the microcirculatory changes in a murine model of sepsis. They reported that stabilization of macrocirculatory
parameters, such as the MAP and cardiac output, was likely to occur at the expense of microcirculatory perfusion (Sand et al., 2015). Therefore, macrocirculatory and microcirculatory changes may not be always in the same extent during injury. During the early management of hemorrhagic shock, fluid resuscitation is often the first modality to restore hemodynamic stability. However, even when systemic
hemodynamic alterations seem restored by fluid resuscitation, considerable alterations in the microcirculation may persist. For example, Legrand et al. reported that despite the restoration of the MAP by fluid resuscitation with 0.9% saline, the fluid
resuscitation does not improve renal microcirculatory oxygenation (Legrand et al., 2010). Our results were consistent with this finding; despite a restoration of the MAP, the microcirculation remained impaired, especially in the intestinal mucosa.
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Traditionally, the fluid requirement was assessed mainly according to the restoration of macrocirculatory parameters, especially the MAP; the fluid volume required to achieve the same MAP target by using a crystalloid instead of a colloid or blood product was considered to be a ratio of 3:1. Currently, the ratio was estimated at approximately 1.5:1 (Orbegozo Cortes et al., 2015) or even less during the acute phase of resuscitation. Our result was consistent with the finding that a lower volume of crystalloid was required to restore the MAP during the acute phase of hemorrhagic shock However, the microcirculation may remain at risk, especially in the intestinal mucosa. Because microcirculatory dysfunction of the intestinal mucosa was related to the disruption of the intestinal barrier caused by bacterial translocation (Zanoni et al., 2009), it can result in multiple organ dysfunction syndrome. The use of additional physiological parameters such as LSCI to monitor the microcirculation during resuscitation may be valuable in effective therapeutic strategies.
We also found that tissue oxygen desaturation among the splanchnic organs after hemorrhagic shock was more homogeneous than the changes in microcirculatory blood flow intensity. Both blood flow and oxygen saturation are equally critical components of microcirculation, but previous studies have insufficiently examined tissue oxygen desaturation after a hemorrhage. Recently, Harrois at el. found that not only hypovolemia but also hypoxemia is detrimental to tissue microcirculation during
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the acute phase of hemorrhagic shock (Harrois et al., 2013). The oxygen consumption of splanchnic organs at rest is approximately 30% of total body oxygen consumption, and they have a large capacity to adapt to reduced blood flow by increasing oxygen extraction (Takala, 1996). We found that tissue oxygen saturation in the splanchnic organs was lower than that in the gracilis muscle after hemorrhagic shock, which may have been due to elevated oxygen extraction. Previous studies have shown that the oxygen supply in the liver is more efficiently maintained than that in other splanchnic organs during minor hemorrhaging until blood loss exceeds 30% (similar to the extent examined in the present study) (Rasmussen et al., 1999, Jakob, 2002). Similarly, our results showed that after severe hemorrhagic shock, hepatic tissue oxygen saturation exhibited a comparable reduction to that in other splanchnic organs. Despite the restoration of the microcirculatory blood flow intensity in the kidney after fluid resuscitation, the tissue oxygenation saturation did not recover because of the lack of oxygen-carrying capability of the resuscitation fluid.
This study had several limitations. First, the microcirculatory blood flow intensity measured by LSCI was derived by the average of the larger ROIs and each animal received the same surgical preparation by the same anatomic landmark (details are mentioned in the supplementary material). Accordingly, bias from sampling variation may be minimized. However, the quantification of microcirculatory blood flow
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intensity may still not be completely precise because small variations in amount of exposed gut indeed present between groups. Therefore, our results regarding the percent change of microcirculatory blood intensity should be more appropriate than the comparison of absolute values. Second, precise fluid replacement for surgical evaporation is sometimes difficult because an overreplacement may underestimate the microcirculatory changes secondary to hemorrhagic shock and an insufficient
replacement may overestimate those changes. Although we subcutaneously injected 10 mL/kg of saline (together with atropine to prevent airway secretion) and the exposed viscera was kept moist with aerosolized prewarmed saline, the long
laparotomy still resulted in significant water evaporation. This may be the reason for the decrease in the intestinal mucosal microcirculatory blood flow intensity in the S group at T1 to T3 (statistically nonsignificant). However, this phenomenon was only evident in the intestinal mucosa; it may be still consistent with our main finding that the intestinal mucosa is the most microcirculatory vulnerable organ among the splanchnic organs. Third, the primary objective of this study was to investigate the microcirculatory changes secondary to hemorrhagic shock among multiple splanchnic organs. The effects of different methods of resuscitation, such as fluid resuscitation by using a colloid or a different fluid volume or by transfusion, were not investigated.
The therapeutic effect of fluid resuscitation after restoration the microcirculation on
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survival was also not investigated. Further research may be warranted and our results may be a valuable reference.
In conclusion, we showed that hemorrhagic shock induced the largest reduction in microcirculatory blood flow intensity in the intestinal mucosa, which also exhibited an unfavorable response to fluid resuscitation despite that the MAP was restored. This model may be applied in future studies investigating the effects of different methods of resuscitation for hemorrhagic shock on microcirculatory changes among multiple splanchnic organs.
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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.
In the current study, we compared commonly used resuscitation fluids, namely a crystalloid (0.9% saline), HTS, and synthetic colloids (GEL and HES), in the acute management of hemorrhagic shock. The major findings are: first, we observed that although fluid resuscitation with the crystalloid restored the MAP and decreased the serum lactatemia, intestinal microcirculation was effectively resuscitated only after using the HTS or synthetic colloids; second, fluid resuscitation using the synthetic colloids was associated with the greatest in vivo renal ROS formation following reperfusion.
Different splanchnic organs may have heterogeneous microcirculatory responses to fluid therapy. For instance, we recently observed that the intestinal microcirculation was more vulnerable to hemorrhaging and had poorer responses to NS resuscitation compared with the liver, kidney, and gracilis muscle (Wu et al., 2015). In support of our previous findings, we also observed that during the resuscitation period, the intestinal microcirculation had a more positive response to fluid resuscitation using HTS, GEL, or HES than to that using NS. Our previous and current studies have indicated that the acute microcirculatory response to fluid therapy in susceptible
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organs such as the intestine is affected not only by volume effects but also by the biochemical composition. Hypertonic fluid and plasma substitutes, especially
synthetic colloids, are the most common compositions for correcting hypovolemia in addition to the crystalloid. HTS was proposed to correct microcirculatory dysfunction and inflammatory effects in a hypovolemic state (Boldt and Ince, 2010) and has been in clinical use for resuscitating hypovolemic and brain injury patients (Strandvik, 2009).It was also reported to improve both macrocirculation and microcirculation in comparison with NS and HES during fluid resuscitation in septic shock patients (van Haren et al., 2012). In previous experimental studies, HTS has improved myocardial blood flow in a pig model of cardiopulmonary resuscitation (Breil et al., 2003), increased cerebral blood flow in a rat model of cardiac arrest (Noppens et al., 2012), and reduced mesenteric microcirculatory dysfunction in a rat model of strangulated small bowel obstruction (Luiz Zanoni et al., 2013). We additionally found that the microcirculatory blood flow in the serosal muscular layer was the most restored in the HTS group (Fig 2D and 3D), probably because HTS is more effective in increasing superior mesenteric arterial blood flow (Brod et al., 2006) and in improving intestinal perfusion with selective vasodilation of precapillary arterioles (Zakaria el et al., 2006) following hemorrhagic shock. Synthetic colloids have been widely used clinically, and their therapeutic effects on microcirculation have gained substantial attention in
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human studies (Boldt and Ince, 2010). GEL was reported to improve splanchnic perfusion in patients who underwent abdominal aortic aneurysm repair (Rittoo et al., 2002) and in hypovolemic septic patients (Asfar et al., 2000). In addition, HES
improved gastric mucosal perfusion in patients who received abdominal aortic surgery (Mahmood et al., 2009) or liver surgery (Cui et al., 2014) and improved sublingual microcirculation during early goal-directed therapy for septic patients (Dubin et al., 2010). However, the effects of synthetic colloids on the microcirculation among multiple splanchnic organs have been less frequently investigated. Our results are in accordance with those of clinical reports indicating that splanchnic microcirculatory blood flow improved after synthetic colloid resuscitation. Synthetic colloids may exert this effect by inducing a decrease in erythrocyte aggregation, thereby reducing the low-shear viscosity of the blood (Neff et al., 2005). Despite the microcirculatory advantages of fluid resuscitation using synthetic colloids, concerns remain regarding the safety of both GEL (Thomas-Rueddel et al., 2012) and HES (Meybohm et al., 2013, Perel et al., 2013), mainly an increased risk of acute kidney injury, especially in critically ill patients who are vulnerable to oxidative stress. Moreover, we determined that increased reperfusion-induced renal ROS formation may be a mechanism
underlying the risk of kidney injury during fluid resuscitation using synthetic colloids.
An ideal resuscitation fluid should not only be effective in restoring both
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macrocirculation and microcirculation but also cause less reperfusion injury (Santry and Alam, 2010). Recently, Chen and colleague reported that fluid resuscitation with HES 130/0.4 following hemorrhagic shock was associated with lesser oxidative stress and a less severe inflammatory response in the liver, intestine, lungs, and brain
compared with GEL and HES 200/0.5 (Chen et al., 2013). By contrast, in the current study, increased formation of renal ROS was evident after fluid resuscitation using GEL and HES for hemorrhagic shock. The differences are likely related to two aspects. First, the comparison among resuscitation fluids was not limited to synthetic colloids in the current study; particularly, HTS was included in the comparison.
Second, the target organ was different. ROS formation in the kidney was emphasized in the current study because infusion of GEL and, particularly, HES is associated with acute kidney injury (Opperer et al., 2015, Perel et al., 2013, Meybohm et al., 2013, Thomas-Rueddel et al., 2012). ROS have an extremely short lifetime, and various antioxidants exist in vivo. Therefore, a general method for detecting the products of lipid peroxidation, such as malondialdehyde, in tissue may be insufficiently sensitive for detecting acute changes in ROS during reperfusion through fluid resuscitation. In the current study, to evaluate ROS production, we used an enhanced
chemiluminescence method that is highly sensitive for detecting acute changes in ROS (Dikalov et al., 2007). Greater reperfusion-induced ROS formation than that
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induced by ischemia may be inevitable after effective microcirculatory restoration;
accordingly, greater ROS formation was observed in the HTS, GEL, and HES groups than in the control and NS groups. However, the higher reperfusion-induced ROS may not be completely explained by more effective microcirculatory restoration, because HTS was comparably effective in restoring splanchnic microcirculation but did not induce higher renal ROS formation than the synthetic colloids did. It may be because the anti-inflammatory properties of HTS. Studies have reported that using HTS was associated with less neutrophil activation (Luiz Zanoni et al., 2013) and less expression of genes implicated in leukocyte–endothelium interaction (van Haren et al., 2011). However, our results should be cautiously applied to clinical scenarios, because there are differences in susceptibility to oxidative challenge between rats and humans (Godin and Garnett, 1992). Rodents may be more resistant to the pathological effects of nitrosative stress, but humans may have evolved counterregulatory
mechanisms (Reade and Young, 2003). Additional investigations may be warranted for understanding the extent of renal ROS formation caused by using synthetic colloids in clinical settings.
In hemorrhagic shock, stabilization of macrocirculatory hemodynamic parameters, such as the MAP, is likely to occur at the expense of splanchnic
microcirculatory perfusion. For instance, Dubin and colleagues reported a dissociation
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between macrocirculation and sublingual as well as intestinal microcirculation during hemorrhaging; the MAP and arterial pH were significantly modified only at the final stage of bleeding, but microcirculation decreased at the first stage of bleeding (Dubin et al., 2009). In the current study, we found that the intestine as the most vulnerable splanchnic tissue during the dissociation between macrocirculation and
microcirculation by conducting a multiple organ model which may be the major difference in the current study comparing to other experimental models. The simultaneously monitoring microcirculation among multiple organs was by using LSCI. The LSCI enables full-field imaging with multiple ROIs and investigating multiple organs in near real time. Furthermore, because a larger ROI can be set, LSCI reduces intersite and interindividual variability and can provide a comparable or even improved reproducibility of microcirculation compared with other techniques, such as sidestream dark-field imaging (Rousseau et al., 2011). However, sidestream dark-field imaging enables direct visual observation of red blood cells flowing through
individual capillaries and can depict heterogeneity between capillaries.
This study has certain limitations. The major limitation is the brief period of
observation because the long laparotomy for exposure of multiple splanchnic organs is associated with significant injury and stress. Therefore it was focused on the period of acute resuscitation, long-term outcomes, such as a survival rate, were less
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appropriate to evaluate. The improvement of outcomes after fluid resuscitation may be associated with other factors in addition to microcirculatory response and
reperfusion injury. For instance, the outcome of resuscitation using HTS was reported to be nonsignificantly more favorable than that of resuscitation using NS in patients receiving out-of-hospital resuscitation for traumatic hemorrhagic shock (Bulger et al., 2011). Second, this study compared the microcirculatory responses of various
splanchnic organs to different resuscitation fluids. Thus, the euvolemic model was applied. Therefore, the results of the current study may not be generalizable to other methods of treatment such as hypotensive resuscitation, which may improve survival following hemorrhagic shock (Santry and Alam, 2010). Third, the microcirculation plays a crucial role in acute kidney injury (Le Dorze et al., 2009). LSCI may enable detecting the heterogeneity in reperfusion dynamics in renal microvascular perfusion (Bezemer et al., 2010), but we did not correlate this heterogeneity with renal ROS formation after synthetic colloid resuscitation in the current study. Because the
primary goal of the current study was to evaluate the microcirculatory changes among multiple splanchnic organs, heterogeneity in a specific single organ was not
examined. However, because the LSCI perfusion distributions of reperfusion
correlated to the changes in the mean value of the entire kidney (Bezemer et al., 2010) and the renal ROI in the current study was close to that of the entire kidney, the
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changes of the mean microcirculatory blood flow presented in this study may still correlate to reperfusion-induced microvascular heterogeneity. Fourth, the current study examined two severities of hemorrhagic shock (30 mL/kg and 20 mL/kg). Most investigators attempt to recreate hemorrhagic shock by inducing blood volume loss of more than 40% (Fulop et al., 2013) because this level of shock is strongly correlated to outcomes. In the current study, this extent of blood loss was reached only in part I of the experiment because volatile anesthesia could not be used during the in vivo ROS measurement. However, it is rational to assume that more reperfusion-induced renal ROS formation occurs when more blood is withdrawn and more fluid is used for resuscitation. Finally, the serum hemoglobin level calculated through arterial blood gas analysis at T2 was higher in the GEL group than in the other groups. Although the values obtained using an arterial blood gas analyzer are reliable for use in detecting serial serum lactatemia changes (Spahn et al., 2013), the obtained hemoglobin values should be interpreted with caution and confirmed using standard venous samples (Quinn et al., 2013).
Conclusions
Our study suggested that even though fluid resuscitation with a crystalloid effectively restored the MAP and decreased serum lactatemia and kidney microcirculation in
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hemorrhagic shock, the intestinal microcirculation was restored only by other volume expanders, 3% HTS and synthetic colloids (GEL and HES). In addition, reperfusion-induced in vivo renal ROS formation was significantly increased in rats resuscitated with the synthetic colloids. Our result supports the clinical literatures that fluid resuscitation may increase the risk of kidney injury.
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Section 2：Predicting stroke volume and arterial pressure fluid responsiveness in liver cirrhosis patients by using dynamic preload variables
In this study, we determined that dynamic preload variables, namely PPV, SVV, and
In this study, we determined that dynamic preload variables, namely PPV, SVV, and