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 that 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 formation of renal ROS in vivo after 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 [8]. 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 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 [1] and has been in clinical use for resuscitating hypovolemic and brain injury patients [18]. It was also reported to improve both macrocirculation and microcirculation in comparison with NS and HES during fluid resuscitation in septic shock patients [19]. In previous experimental studies, HTS has improved myocardial blood flow in a pig model of cardiopulmonary resuscitation [20], increased cerebral blood flow in a rat model of cardiac arrest [21], and reduced mesenteric microcirculatory dysfunction in a rat model of strangulated small bowel obstruction [22]. We additionally found that the microcirculatory blood flow in the serosal muscular layer was the most restored in the HTS group (Figs. 2d and 3d), probably because HTS is more effective in increasing superior mesenteric arterial blood flow [23] and in improving intestinal perfusion with selective vasodilation of precapillary arterioles [24] after hemorrhagic shock. Synthetic colloids have been widely used clinically, and their therapeutic effects on microcirculation have gained substantial attention in human studies [1]. GEL was reported to improve splanchnic perfusion in patients who underwent abdominal aortic aneurysm repair [25] and in hypovolemic septic patients [26]. In addition, HES improved gastric mucosal perfusion in patients who received abdominal aortic surgery [27] or liver surgery [28] and improved sublingual microcirculation during early goal-directed therapy for septic patients [29]. 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 [30]. Despite the microcirculatory advantages of fluid resuscitation using synthetic colloids, concerns remain about the safety of both GEL [13] and HES [11, 12], 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 macrocirculation and microcirculation but also cause less reperfusion injury [31]. Recently, Chen and colleague reported that fluid resuscitation with HES 130/0.4 after 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 [32]. 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 [11‐13, 33]. ROS have an extremely short lifetime, and there are various antioxidants 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 CL method that is highly sensitive for detecting acute changes in ROS [34]. Greater reperfusion-induced ROS formation than that 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. It may be because of the anti-inflammatory properties of HTS. Studies have reported that using HTS is associated with less neutrophil activation [22] and less expression of genes implicated in leukocyte–endothelium interaction [35]. However, our results should be cautiously applied to clinical scenarios, because there are differences in susceptibility to oxidative challenge between rats and humans [36]. Rodents may be more resistant to the pathological effects of nitrosative stress, but humans may have evolved counter-regulatory mechanisms [37]. 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 between macrocirculation, sublingual and 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 [38]. In the current study, we found that the intestine was 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 compared to other experimental models. The simultaneously monitoring of microcirculation among multiple organs was performed 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 inter-site and inter-individual variability and can provide comparable or even improved reproducibility of microcirculation compared with other techniques, such as sidestream dark-field imaging [39]. 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 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 non-significantly more favorable than that of resuscitation using NS in patients receiving out-of-hospital resuscitation for traumatic hemorrhagic shock [40]. 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 after hemorrhagic shock [31]. Third, the microcirculation plays a crucial role in acute kidney injury [41]. LSCI may enable detection of the heterogeneity in reperfusion dynamics in renal microvascular perfusion [42], 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 [42] and the renal ROI in the current study was close to that of the entire kidney, the changes in 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 % [43] 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 [44], the obtained hemoglobin values should be interpreted with caution and confirmed using standard venous samples [45].