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Laser speckle contrast imaging for assessing microcirculatory changes in multiple splanchnic organs and the gracilis muscle during hemorrhagic shock and fluid resuscitation
Microvascular Research 101 (2015) 55–61
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Laser speckle contrast imaging for assessing microcirculatory changes in multiple splanchnic organs and the gracilis muscle during hemorrhagic shock and fluid resuscitation Chun-Yu Wu a,b, Yu-Chang Yeh a, Chiang-Ting Chien b, Anne Chao a, Wei-Zen Sun a,⁎, Ya-Jung Cheng a,⁎, on behalf of the NTUH Center of Microcirculation Medical Research (NCMMR) a b
Department of Anesthesiology, National Taiwan University Hospital, Taipei, Taiwan Department of Life Science, National Taiwan Normal University, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Accepted 6 June 2015 Available online 17 June 2015 Keywords: Hemorrhagic shock Microcirculation Multiple splanchnic organs Laser speckle contrast imaging Fluid resuscitation
a b s t r a c t Objective: Hemorrhagic shock induces both macrocirculatory and microcirculatory impairment. Persistent microcirculatory dysfunction is associated with the dysfunction of multiple organs, especially in the splanchnic organs. However, few studies have simultaneously investigated microcirculation in multiple organs. In the present study, we used laser speckle contrast imaging to simultaneously investigate microcirculatory changes secondary to hemorrhagic shock and after fluid resuscitation among multiple splanchnic organs and the gracilis muscle. Materials and methods: 72 male Wistar rats were subjected to sham operation, hemorrhagic shock (total blood loss of 30 mL/kg) and saline resuscitation. Macrocirculatory parameters, including the mean arterial pressure (MAP) and heart rate, and microcirculatory parameters, including microcirculatory blood flow intensity and tissue oxygen saturation in the liver, kidney, intestine (mucosa, serosal muscular layer, and Peyer's patch), and gracilis muscle were compared in a period of 3 h. Results: Hemorrhagic shock induced a significant reduction of microcirculatory blood flow intensity in the kidney and intestine (especially the mucosa). Tissue oxygen saturation reduction secondary to hemorrhagic shock was comparable among the various splanchnic organs but lower than the gracilis muscle. Fluid resuscitation restored the MAP but not the microcirculatory blood flow in the intestine and the tissue oxygen saturation in each splanchnic organ. Conclusion: Hemorrhagic shock induced the largest reduction in microcirculatory blood flow intensity in the intestinal mucosa. By comparison, the reduction of tissue oxygen saturation was not significantly different among the various splanchnic organs. Although fluid resuscitation restored the MAP, the intestinal microcirculation remained damaged. © 2015 Elsevier Inc. All rights reserved.
Introduction Hemorrhagic shock is one of the major causes of mortality in traumatic injury. Both macrocirculatory and microcirculatory dysfunctions have been characterized in the acute phase of hemorrhagic shock (Dubin et al., 2009; van Iterson et al., 2012). Recently, there is a paradigm shift from macrocirculatory to microcirculatory investigations because the persistence of microcirculatory dysfunction is associated with organ failure. During a hemorrhage, blood flow and tissue oxygenation of nonvital organs decrease to maintain the circulation required by vital organs. However, the acute changes of microcirculation blood flow and tissue oxygen saturation in multiple splanchnic organs secondary to hemorrhagic shock are insufficiently clear. Understanding the microcirculatory changes of splanchnic organs during a hemorrhage and ⁎ Corresponding authors. E-mail addresses: [email protected] (W.-Z. Sun), [email protected] (Y.-J. Cheng).
http://dx.doi.org/10.1016/j.mvr.2015.06.003 0026-2862/© 2015 Elsevier Inc. All rights reserved.
resuscitation may provide crucial information for further research and treatment because splanchnic ischemia is one of the major causes of multiple organ dysfunction syndrome (Pastores et al., 1996). Laser speckle contrast imaging (LSCI) is an increasingly prevalent technique for monitoring microcirculatory blood flow. Because it enables full-field imaging in near real time with multiple regions of interest, it is suitable for investigating microcirculatory changes among multiple organs (Boas and Dunn, 2010; Ding et al., 2014; Draijer et al., 2009). LSCI in combination with tissue oxygen saturation measurements may offer a comprehensive understanding of acute microcirculatory changes among multiple organs. In this study, we investigated the heterogeneity of microcirculatory responses to hemorrhagic shock among multiple splanchnic organs and the gracilis muscle by using LSCI and tissue oxygen saturation measurements. Additionally, we also clarified the microcirculatory effects of fluid resuscitation during the acute phase of hemorrhagic shock. Because different tissues of the intestine may have different susceptibilities to shock (Yeh et al.,
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2012b), further investigation was also conducted to compare the microcirculatory changes of the intestinal mucosa, serosal muscular layer, and lymph node.
for examining the microcirculation. Moreover, the right gracilis muscle was exposed for measuring microcirculatory changes relative to those of the splanchnic organs. The exposed viscera and tissue were kept moist hourly with saline (0.5 mL of aerosolized) prewarmed to 37 °C.
Materials and methods Hemorrhagic shock and fluid resuscitation protocol Experimental animals A total of 72 male Wistar rats were used (body weight 250 ± 50 g; Biolasco Taiwan Co., Taipei, Taiwan). The rats were kept on a 12-h light–dark cycle and had free access to water and food. 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., 2012b). A tracheostomy was performed, and 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. 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 body 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 macrocirculatory hemodynamics, including the mean arterial pressure (MAP) and heart rate (HR), and to withdraw blood to establish hemorrhagic shock. The external jugular vein was used for infusion of resuscitation fluid. A long midline laparotomy was performed to exteriorize splanchnic organs including the liver, left kidney, and a segment of the terminal ileum (about 6 to 10 cm proximal to the ileocecal valve). A 2-cm section was performed 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 examining the microcirculation. 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
After completion of the surgery, the animals were allowed to stabilize for 30 min before the baseline measurements were performed (baseline condition was considered stable when all measurement values remained at 10% for 15 min; T0). After the baseline 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 time points shown in Fig. 1. In the S group, the rats received the same surgical preparation but did not undergo 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 in the splanchnic organs (Yeh et al., 2012a). The detail of choices of interested region of tissue for monitoring is mentioned in the Supplementary Material. 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 is related to high flow and high contrast to 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
Fig. 1. Timeline of protocol.
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Table 1 Macrocirculatory changes secondary to hemorrhagic shock.
MAP (mm Hg) HR (bpm)
S H R S H R
T0 baseline
T1 1 h after shock
T2 2 h after shock
T3 3 h after shock
ANOVA group
ANOVA time
ANOVA interaction
105 ± 5 108 ± 5 102 ± 4 376 ± 12 391 ± 17 334 ± 9
109 ± 5 68 ± 9⁎# 91 ± 7# 389 ± 19 364 ± 16 336 ± 15
99 ± 3 66 ± 6⁎# 84 ± 7# 370 ± 20 377 ± 15 348 ± 18
97 ± 9 60 ± 5⁎# 85 ± 5# 359 ± 16 386 ± 16 359 ± 14
P = 0.005
p b 0.001
P b 0.001
p = 0.200
P = 0.712
P = 0.105
MAP, mean arterial pressure; HR, heart rate (beats per minute). S, sham operation group; H, hemorrhagic shock group; R, resuscitation group. ⁎ p b 0.05 compared with sham. # p b 0.05 compared with baseline value.
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. 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 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. 1). 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 values of microcirculatory blood flow intensity and 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. Statistical analysis The results are presented as the mean ± standard error. Two-way repeated measures of ANOVA followed by Tukey test as a post test (nonparametric data was transformed in the ranks first) were used to analyze and compare sequential changes in the (1) absolute values of the MAP, HR, microcirculatory blood flow intensity, and oxygen saturation in individual organs at the designated time points and (2) percentages of microcirculatory blood flow intensity and oxygen saturation from the baseline values of the various organs. A P value lower than 0.05 was considered statistically significant. All statistical analyses were performed and all graphs were plotted using SigmaPlot for Windows Version 12 (SAS Institute, Cary, NC, USA). Results Macrocirculatory changes secondary to hemorrhagic shock The MAP significantly decreased after hemorrhagic shock (MAP at T1, T2, and T3 was significantly lower than that at T0; p b 0.001), whereas there was no significant MAP change in the S group (Table 1). After fluid resuscitation, the MAP at T1 to T3 was comparable in the S group (Table 1). The HR did not significantly change in each group.
Table 2 Splanchnic organ microcirculatory blood flow intensity changes secondary to hemorrhagic shock. Flux (PU) Liver
Kidney
Intestinal mucosa
Serosal muscular layer
Peyer's patch
Gracilis muscle
S H R S H R S H R S H R S H R S H R
T0
T1
T2
T3
ANOVA group
ANOVA time
ANOVA interaction
1162 ± 81 1131 ± 54 1259 ± 52 1598 ± 83 1500 ± 74 1660 ± 52 1352 ± 154 1539 ± 119 1826 ± 119⁎ 943 ± 58 980 ± 123 1062 ± 61 1533 ± 195 1335 ± 204 1613 ± 123 947 ± 43 1013 ± 121 1025 ± 65
1120 ± 82 913 ± 90# 1269 ± 74 1526 ± 102 939 ± 80⁎#
1119 ± 50 977 ± 99 1195 ± 54 1563 ± 96 1121 ± 85⁎#
p = 0.045
p = 0.115
P = 0.341
p = 0.002
p b 0.001
p b 0.001
1546 ± 98 1326 ± 90 534 ± 73⁎# 819 ± 67⁎# 1088 ± 86 565 ± 116⁎# 892 ± 64# 1528 ± 170 579 ± 51⁎#
1499 ± 77 1169 ± 71 433 ± 35⁎#
1032 ± 53 962 ± 120 1219 ± 62 1554 ± 96 1047 ± 124⁎# 1512 ± 87 1094 ± 59 457 ± 18⁎#
p b 0.001
p b 0.001
p b 0.001
p = 0.008
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p = 0.078
p = 0.314
p = 0.052
1230 ± 82 1122 ± 143 812 ± 88⁎ 1137 ± 97
S, sham operation group; H, hemorrhagic shock group; PU, perfusion unit. ⁎ p b 0.05 compared with sham. # p b 0.05 compared with baseline value.
843 ± 100⁎# 1109 ± 104 568 ± 73⁎# 766 ± 33⁎# 1464 ± 173 625 ± 55⁎# 1087 ± 63# 1104 ± 133 744 ± 47⁎ 1062 ± 70
822 ± 100# 916 ± 134 519 ± 122⁎# 688 ± 64# 1300 ± 161 564 ± 47⁎# 960 ± 76# 1058 ± 136 739 ± 48⁎ 957 ± 96
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Splanchnic organs and gracilis muscle microcirculatory blood flow intensity changes secondary to hemorrhagic shock and fluid resuscitation Table 2 shows a summary of the sequential absolute value changes in microcirculatory blood flow intensity during the measurement period. The sham operation induced no significant changes in microcirculatory blood flow intensity in each investigated organs (Figs. 3B and E). By comparison, hemorrhagic shock induced a significant reduction in microcirculatory blood flow intensity in the kidney and intestine (including the mucosa, serosal muscular layer, and Peyer's patch). Hemorrhagic shock induced heterogeneous sequential percent changes in microcirculatory blood flow intensity, with the following reduction differences: intestinal mucosa N serosal muscular layer, Peyer's patch N liver, kidney, and gracilis muscle (p b 0.05; Figs. 2A, C, and E; Figs. 3C and F). Compared to the H group, fluid resuscitation significantly improved the microcirculatory blood flow intensity in the splanchnic organ at T1 (Fig. 2A), but only restored the microcirculatory blood flow intensity impairment of the kidney and partially restored the
microcirculatory blood flow intensity impairment of intestinal mucosa at T3 (Figs. 2E, 3F, and G). After fluid resuscitation, the intestinal microcirculatory blood flow intensity remained compromised compared to the baseline condition, especially in the mucosa (Table 2; Figs. 2E and 3G).
Splanchnic organs and gracilis muscle tissue oxygen saturation changes secondary to hemorrhagic shock and fluid resuscitation Table 3 shows a summary of the sequential absolute value changes in tissue oxygen saturation during hemorrhagic shock. There was no significant tissue oxygen saturation change among the organs measured in the S group. Hemorrhagic shock induced significant homogeneous changes of tissue oxygen saturation in each investigated target organ; the reductions in the tissue oxygen saturation of the various splanchnic organs were comparable but significantly greater than those of the gracilis muscle (p b 0.05; Figs. 2B, D, and F). Compared to the H group,
Fig. 2. (A–F) Percent changes of microcirculatory blood flow intensity (2A, 2C, and 2E) and tissue oxygen saturation (2B, 2D, and 2F) at T1, T2, and T3 compared with T0 (2A and 2B: T1 vs. T0; 2C and 2D: T2 vs. T0; and 2E and 2F: T3 vs. T0). Blank bars with bold edges represent changes among the splanchnic organs of the H group. Bars with a grey slash represent changes among the splanchnic organs of the R group. The groups marked with 1, 2, and 3 have differing average percent changes of microcirculatory blood flow intensity (1 b 2 b 3, p b 0.05). The groups marked with ^ represent a significant change between the H and R groups.
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fluid resuscitation did not significantly improve tissue oxygen saturation (Figs. 2B, D, and F). Discussion In this study, we examined a rat model that could 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
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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; and (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
Fig. 3. (A–G) Example of an exposed viscera (Fig. 3A) and laser speckle contrast imaging of the microcirculatory blood flow intensities in the S (3B and 3E), H (3C and 3F), and R (3D and 3G) groups at T0 and T3. The regions of interest were marked as liver (1), kidney (2), intestinal mucosa (3), serosal muscular layer (4), Peyer's patch (5), and gracilis muscle (6).
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time; therefore, it enables simultaneous measurement of changes in microcirculatory blood flow intensity among multiple organs (Beilman et al., 1999; Morini et al., 2000; Sordia et al., 2004; van Iterson et al., 2012). 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 a comparable or even improved reproducibility of microcirculation in comparison with other techniques such as laser Doppler flowmetry or sidestream dark-field (SDF) imaging (Rousseau et al., 2011; Roustit et al., 2010; Sturesson et al., 2013; Tew 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 et al. found that the intestine was more sensitive to hemorrhaging than the heart in a pig model of hemorrhagic shock (Vajda et al., 2004). Additionally, we found that, with heterogeneous impairment of microcirculatory blood flow intensity within the 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 hemodynamics, 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. 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 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) (Jakob, 2002; Rasmussen et al., 1999). 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
Table 3 Splanchnic organ tissue oxygen saturation changes secondary to hemorrhagic shock. StO2 (%) Liver
Kidney
Intestinal mucosa
Serosal muscular layer
Peyer's patch
Gracilis muscle
S H R S H R S H R S H R S H R S H R
T0
T1
T2
T3
ANOVA group
ANOVA time
ANOVA interaction
66.4 ± 2.5 63.9 ± 1.0 57.8 ± 2.4 77.5 ± 1.5 70.1 ± 1.4 71.5 ± 3.0 56.8 ± 1.5 55.8 ± 3.2 57.0 ± 2.0 63.5 ± 2.0 68.2 ± 2.2 66.7 ± 2.8 58.8 ± 1.9 64.8 ± 3.3 65.0 ± 1.3 40.7 ± 3.0 55.2 ± 2.0⁎ 43.2 ± 4.2
69.9 ± 0.6 14.7 ± 3.4⁎# 16.7 ± 5.4⁎# 79.53 ± 1.7 29.2 ± 4.2⁎# 32.8 ± 7.4⁎#
68.03 ± 2.4 38.0 ± 4.0⁎# 39.2 ± 5.6⁎# 76.6 ± 1.7 50.5 ± 2.7⁎# 56.1 ± 2.9⁎#
64.18 ± 2.5 45.8 ± 7.1⁎# 45.8 ± 4.7⁎# 79.8 ± 1.6 54.0 ± 2.9⁎# 56.2 ± 5.5⁎#
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
53.2 ± 2.1 10.1 ± 3.8⁎# 4.3 ± 2.2⁎# 58.6 ± 2.9 18.0 ± 3.5⁎# 11.0 ± 4.7⁎#
49.9 ± 1.9 24.4 ± 4.9⁎# 29.1 ± 3.7⁎# 59.2 ± 0.9 39.2 ± 3.2⁎# 46.0 ± 4.9⁎#
50.5 ± 2.2 34.1 ± 2.7⁎# 31.4 ± 3.8⁎# 59.6 ± 2.1 46.2 ± 4.5⁎# 44.0 ± 4.2⁎#
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
57.6 ± 1.8 27.8 ± 6.1⁎# 19.2 ± 5.1⁎#
59.0 ± 1.5 46.3 ± 3.6⁎# 54.3 ± 4.1# 39.4 ± 2.1 38.7 ± 2.0# 28.5 ± 1.7#
60.1 ± 1.5 46.0 ± 3.3⁎# 50.9 ± 3.3⁎# 45.2 ± 2.5 45.8 ± 4.6 38.0 ± 2.8
p = 0.008
p b 0.001
p b 0.001
p = 0.016
p b 0.001
p b 0.001
44.4 ± 2.2 31.1 ± 4.6⁎# 21.3 ± 6.2⁎#
S, sham operation group; H, hemorrhagic shock group; StO2, tissue oxygen saturation. ⁎ p b 0.05 compared with sham. # p b 0.05 compared with baseline value.
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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 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 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. We suggest that this model be applied in future studies investigating the effects of different methods of resuscitation for hemorrhagic shock on microcirculatory changes among splanchnic organs. Acknowledgments This study was supported in part by the research grant NTUH.103S2361 from National Taiwan University Hospital. We thank Zong-Gin Wu (Technician, Department of Surgery, National Taiwan University Hospital) and Roger Lien (Technician, MicroStar Instruments Co., Ltd., Taipei, Taiwan) for their technical assistance.
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Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mvr.2015.06.003. References Beilman, G.J., et al., 1999. Near-infrared spectroscopy measurement of regional tissue oxyhemoglobin saturation during hemorrhagic shock. Shock 12, 196–200. Boas, D.A., Dunn, A.K., 2010. Laser speckle contrast imaging in biomedical optics. J. Biomed. Opt. 15, 011109. Ding, C., et al., 2014. Laser speckle contrast imaging for assessment of abdominal visceral microcirculation in acute peritonitis: does sequential impairments exist? Microvasc. Res. 95, 26–30. Draijer, M., et al., 2009. Review of laser speckle contrast techniques for visualizing tissue perfusion. Lasers Med. Sci. 24, 639–651. Dubin, A., et al., 2009. Systemic and microcirculatory responses to progressive hemorrhage. Intensive Care Med. 35, 556–564. Harrois, A., et al., 2013. Synergistic deleterious effect of hypoxemia and hypovolemia on microcirculation in intestinal villi*. Crit. Care Med. 41, e376–e384. Jakob, S.M., 2002. Clinical review: splanchnic ischaemia. Crit. Care 6, 306–312. Legrand, M., et al., 2010. Fluid resuscitation does not improve renal oxygenation during hemorrhagic shock in rats. Anesthesiology 112, 119–127. Liu, H., et al., 2011. Design of a tissue oxygenation monitor and verification on human skin. In: Ramanujam, N., Popp, J. (Eds.), Clinical and Biomedical Spectroscopy and Imaging II Vol. 8087. Optical Society of America, Munich, p. 80871Y. Morini, S., et al., 2000. Intestinal microvascular patterns during hemorrhagic shock. Dig. Dis. Sci. 45, 710–722. Orbegozo Cortes, D., et al., 2015. Crystalloids versus colloids: exploring differences in fluid requirements by systematic review and meta-regression. Anesth. Analg. 120, 389–402. Pastores, S.M., et al., 1996. Splanchnic ischemia and gut mucosal injury in sepsis and the multiple organ dysfunction syndrome. Am. J. Gastroenterol. 91, 1697–1710. Rasmussen, A., et al., 1999. Preserved arterial flow secures hepatic oxygenation during haemorrhage in the pig. J. Physiol. 516 (Pt 2), 539–548. Rousseau, P., et al., 2011. Increasing the “region of interest” and “time of interest”, both reduce the variability of blood flow measurements using laser speckle contrast imaging. Microvasc. Res. 82, 88–91. Roustit, M., et al., 2010. Excellent reproducibility of laser speckle contrast imaging to assess skin microvascular reactivity. Microvasc. Res. 80, 505–511. Sand, C.A., et al., 2015. Quantification of microcirculatory blood flow: a sensitive and clinically relevant prognostic marker in murine models of sepsis. J. Appl. Physiol. 118, 344–354 (1985). Sordia, T., et al., 2004. Hemorheological disorders in the microcirculation following hemorrhage. Clin. Hemorheol. Microcirc. 30, 461–462. Sturesson, C., et al., 2013. Laser speckle contrast imaging for assessment of liver microcirculation. Microvasc. Res. 87, 34–40. Takala, J., 1996. Determinants of splanchnic blood flow. Br. J. Anaesth. 77, 50–58. Tew, G.A., et al., 2011. Comparison of laser speckle contrast imaging with laser Doppler for assessing microvascular function. Microvasc. Res. 82, 326–332. Vajda, K., et al., 2004. Microcirculatory heterogeneity in the rat small intestine during compromised flow conditions. Microcirculation 11, 307–315. van Iterson, M., et al., 2012. Microcirculation follows macrocirculation in heart and gut in the acute phase of hemorrhagic shock and isovolemic autologous whole blood resuscitation in pigs. Transfusion 52, 1552–1559. Yeh, Y.C., et al., 2012a. Dexmedetomidine prevents alterations of intestinal microcirculation that are induced by surgical stress and pain in a novel rat model. Anesth. Analg. 115, 46–53. Yeh, Y.C., et al., 2012b. Enoxaparin sodium prevents intestinal microcirculatory dysfunction in endotoxemic rats. Crit. Care 16, R59. Zanoni, F.L., et al., 2009. Mesenteric microcirculatory dysfunctions and translocation of indigenous bacteria in a rat model of strangulated small bowel obstruction. Clinics (Sao Paulo) 64, 911–919.
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Laser speckle contrast imaging for assessing microcirculatory changes in multiple splanchnic organs and the gracilis muscle during hemorrhagic shock and fluid resuscitation Chun-Yu Wu a,b, Yu-Chang Yeh a, Chiang-Ting Chien b, Anne Chao a, Wei-Zen Sun a,⁎, Ya-Jung Cheng a,⁎, on behalf of the NTUH Center of Microcirculation Medical Research (NCMMR) a b
Department of Anesthesiology, National Taiwan University Hospital, Taipei, Taiwan Department of Life Science, National Taiwan Normal University, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Accepted 6 June 2015 Available online 17 June 2015 Keywords: Hemorrhagic shock Microcirculation Multiple splanchnic organs Laser speckle contrast imaging Fluid resuscitation
a b s t r a c t Objective: Hemorrhagic shock induces both macrocirculatory and microcirculatory impairment. Persistent microcirculatory dysfunction is associated with the dysfunction of multiple organs, especially in the splanchnic organs. However, few studies have simultaneously investigated microcirculation in multiple organs. In the present study, we used laser speckle contrast imaging to simultaneously investigate microcirculatory changes secondary to hemorrhagic shock and after fluid resuscitation among multiple splanchnic organs and the gracilis muscle. Materials and methods: 72 male Wistar rats were subjected to sham operation, hemorrhagic shock (total blood loss of 30 mL/kg) and saline resuscitation. Macrocirculatory parameters, including the mean arterial pressure (MAP) and heart rate, and microcirculatory parameters, including microcirculatory blood flow intensity and tissue oxygen saturation in the liver, kidney, intestine (mucosa, serosal muscular layer, and Peyer's patch), and gracilis muscle were compared in a period of 3 h. Results: Hemorrhagic shock induced a significant reduction of microcirculatory blood flow intensity in the kidney and intestine (especially the mucosa). Tissue oxygen saturation reduction secondary to hemorrhagic shock was comparable among the various splanchnic organs but lower than the gracilis muscle. Fluid resuscitation restored the MAP but not the microcirculatory blood flow in the intestine and the tissue oxygen saturation in each splanchnic organ. Conclusion: Hemorrhagic shock induced the largest reduction in microcirculatory blood flow intensity in the intestinal mucosa. By comparison, the reduction of tissue oxygen saturation was not significantly different among the various splanchnic organs. Although fluid resuscitation restored the MAP, the intestinal microcirculation remained damaged. © 2015 Elsevier Inc. All rights reserved.
Introduction Hemorrhagic shock is one of the major causes of mortality in traumatic injury. Both macrocirculatory and microcirculatory dysfunctions have been characterized in the acute phase of hemorrhagic shock (Dubin et al., 2009; van Iterson et al., 2012). Recently, there is a paradigm shift from macrocirculatory to microcirculatory investigations because the persistence of microcirculatory dysfunction is associated with organ failure. During a hemorrhage, blood flow and tissue oxygenation of nonvital organs decrease to maintain the circulation required by vital organs. However, the acute changes of microcirculation blood flow and tissue oxygen saturation in multiple splanchnic organs secondary to hemorrhagic shock are insufficiently clear. Understanding the microcirculatory changes of splanchnic organs during a hemorrhage and ⁎ Corresponding authors. E-mail addresses: [email protected] (W.-Z. Sun), [email protected] (Y.-J. Cheng).
http://dx.doi.org/10.1016/j.mvr.2015.06.003 0026-2862/© 2015 Elsevier Inc. All rights reserved.
resuscitation may provide crucial information for further research and treatment because splanchnic ischemia is one of the major causes of multiple organ dysfunction syndrome (Pastores et al., 1996). Laser speckle contrast imaging (LSCI) is an increasingly prevalent technique for monitoring microcirculatory blood flow. Because it enables full-field imaging in near real time with multiple regions of interest, it is suitable for investigating microcirculatory changes among multiple organs (Boas and Dunn, 2010; Ding et al., 2014; Draijer et al., 2009). LSCI in combination with tissue oxygen saturation measurements may offer a comprehensive understanding of acute microcirculatory changes among multiple organs. In this study, we investigated the heterogeneity of microcirculatory responses to hemorrhagic shock among multiple splanchnic organs and the gracilis muscle by using LSCI and tissue oxygen saturation measurements. Additionally, we also clarified the microcirculatory effects of fluid resuscitation during the acute phase of hemorrhagic shock. Because different tissues of the intestine may have different susceptibilities to shock (Yeh et al.,
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2012b), further investigation was also conducted to compare the microcirculatory changes of the intestinal mucosa, serosal muscular layer, and lymph node.
for examining the microcirculation. Moreover, the right gracilis muscle was exposed for measuring microcirculatory changes relative to those of the splanchnic organs. The exposed viscera and tissue were kept moist hourly with saline (0.5 mL of aerosolized) prewarmed to 37 °C.
Materials and methods Hemorrhagic shock and fluid resuscitation protocol Experimental animals A total of 72 male Wistar rats were used (body weight 250 ± 50 g; Biolasco Taiwan Co., Taipei, Taiwan). The rats were kept on a 12-h light–dark cycle and had free access to water and food. 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., 2012b). A tracheostomy was performed, and 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. 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 body 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 macrocirculatory hemodynamics, including the mean arterial pressure (MAP) and heart rate (HR), and to withdraw blood to establish hemorrhagic shock. The external jugular vein was used for infusion of resuscitation fluid. A long midline laparotomy was performed to exteriorize splanchnic organs including the liver, left kidney, and a segment of the terminal ileum (about 6 to 10 cm proximal to the ileocecal valve). A 2-cm section was performed 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 examining the microcirculation. 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
After completion of the surgery, the animals were allowed to stabilize for 30 min before the baseline measurements were performed (baseline condition was considered stable when all measurement values remained at 10% for 15 min; T0). After the baseline 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 time points shown in Fig. 1. In the S group, the rats received the same surgical preparation but did not undergo 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 in the splanchnic organs (Yeh et al., 2012a). The detail of choices of interested region of tissue for monitoring is mentioned in the Supplementary Material. 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 is related to high flow and high contrast to 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
Fig. 1. Timeline of protocol.
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Table 1 Macrocirculatory changes secondary to hemorrhagic shock.
MAP (mm Hg) HR (bpm)
S H R S H R
T0 baseline
T1 1 h after shock
T2 2 h after shock
T3 3 h after shock
ANOVA group
ANOVA time
ANOVA interaction
105 ± 5 108 ± 5 102 ± 4 376 ± 12 391 ± 17 334 ± 9
109 ± 5 68 ± 9⁎# 91 ± 7# 389 ± 19 364 ± 16 336 ± 15
99 ± 3 66 ± 6⁎# 84 ± 7# 370 ± 20 377 ± 15 348 ± 18
97 ± 9 60 ± 5⁎# 85 ± 5# 359 ± 16 386 ± 16 359 ± 14
P = 0.005
p b 0.001
P b 0.001
p = 0.200
P = 0.712
P = 0.105
MAP, mean arterial pressure; HR, heart rate (beats per minute). S, sham operation group; H, hemorrhagic shock group; R, resuscitation group. ⁎ p b 0.05 compared with sham. # p b 0.05 compared with baseline value.
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. 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 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. 1). 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 values of microcirculatory blood flow intensity and 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. Statistical analysis The results are presented as the mean ± standard error. Two-way repeated measures of ANOVA followed by Tukey test as a post test (nonparametric data was transformed in the ranks first) were used to analyze and compare sequential changes in the (1) absolute values of the MAP, HR, microcirculatory blood flow intensity, and oxygen saturation in individual organs at the designated time points and (2) percentages of microcirculatory blood flow intensity and oxygen saturation from the baseline values of the various organs. A P value lower than 0.05 was considered statistically significant. All statistical analyses were performed and all graphs were plotted using SigmaPlot for Windows Version 12 (SAS Institute, Cary, NC, USA). Results Macrocirculatory changes secondary to hemorrhagic shock The MAP significantly decreased after hemorrhagic shock (MAP at T1, T2, and T3 was significantly lower than that at T0; p b 0.001), whereas there was no significant MAP change in the S group (Table 1). After fluid resuscitation, the MAP at T1 to T3 was comparable in the S group (Table 1). The HR did not significantly change in each group.
Table 2 Splanchnic organ microcirculatory blood flow intensity changes secondary to hemorrhagic shock. Flux (PU) Liver
Kidney
Intestinal mucosa
Serosal muscular layer
Peyer's patch
Gracilis muscle
S H R S H R S H R S H R S H R S H R
T0
T1
T2
T3
ANOVA group
ANOVA time
ANOVA interaction
1162 ± 81 1131 ± 54 1259 ± 52 1598 ± 83 1500 ± 74 1660 ± 52 1352 ± 154 1539 ± 119 1826 ± 119⁎ 943 ± 58 980 ± 123 1062 ± 61 1533 ± 195 1335 ± 204 1613 ± 123 947 ± 43 1013 ± 121 1025 ± 65
1120 ± 82 913 ± 90# 1269 ± 74 1526 ± 102 939 ± 80⁎#
1119 ± 50 977 ± 99 1195 ± 54 1563 ± 96 1121 ± 85⁎#
p = 0.045
p = 0.115
P = 0.341
p = 0.002
p b 0.001
p b 0.001
1546 ± 98 1326 ± 90 534 ± 73⁎# 819 ± 67⁎# 1088 ± 86 565 ± 116⁎# 892 ± 64# 1528 ± 170 579 ± 51⁎#
1499 ± 77 1169 ± 71 433 ± 35⁎#
1032 ± 53 962 ± 120 1219 ± 62 1554 ± 96 1047 ± 124⁎# 1512 ± 87 1094 ± 59 457 ± 18⁎#
p b 0.001
p b 0.001
p b 0.001
p = 0.008
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p = 0.078
p = 0.314
p = 0.052
1230 ± 82 1122 ± 143 812 ± 88⁎ 1137 ± 97
S, sham operation group; H, hemorrhagic shock group; PU, perfusion unit. ⁎ p b 0.05 compared with sham. # p b 0.05 compared with baseline value.
843 ± 100⁎# 1109 ± 104 568 ± 73⁎# 766 ± 33⁎# 1464 ± 173 625 ± 55⁎# 1087 ± 63# 1104 ± 133 744 ± 47⁎ 1062 ± 70
822 ± 100# 916 ± 134 519 ± 122⁎# 688 ± 64# 1300 ± 161 564 ± 47⁎# 960 ± 76# 1058 ± 136 739 ± 48⁎ 957 ± 96
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Splanchnic organs and gracilis muscle microcirculatory blood flow intensity changes secondary to hemorrhagic shock and fluid resuscitation Table 2 shows a summary of the sequential absolute value changes in microcirculatory blood flow intensity during the measurement period. The sham operation induced no significant changes in microcirculatory blood flow intensity in each investigated organs (Figs. 3B and E). By comparison, hemorrhagic shock induced a significant reduction in microcirculatory blood flow intensity in the kidney and intestine (including the mucosa, serosal muscular layer, and Peyer's patch). Hemorrhagic shock induced heterogeneous sequential percent changes in microcirculatory blood flow intensity, with the following reduction differences: intestinal mucosa N serosal muscular layer, Peyer's patch N liver, kidney, and gracilis muscle (p b 0.05; Figs. 2A, C, and E; Figs. 3C and F). Compared to the H group, fluid resuscitation significantly improved the microcirculatory blood flow intensity in the splanchnic organ at T1 (Fig. 2A), but only restored the microcirculatory blood flow intensity impairment of the kidney and partially restored the
microcirculatory blood flow intensity impairment of intestinal mucosa at T3 (Figs. 2E, 3F, and G). After fluid resuscitation, the intestinal microcirculatory blood flow intensity remained compromised compared to the baseline condition, especially in the mucosa (Table 2; Figs. 2E and 3G).
Splanchnic organs and gracilis muscle tissue oxygen saturation changes secondary to hemorrhagic shock and fluid resuscitation Table 3 shows a summary of the sequential absolute value changes in tissue oxygen saturation during hemorrhagic shock. There was no significant tissue oxygen saturation change among the organs measured in the S group. Hemorrhagic shock induced significant homogeneous changes of tissue oxygen saturation in each investigated target organ; the reductions in the tissue oxygen saturation of the various splanchnic organs were comparable but significantly greater than those of the gracilis muscle (p b 0.05; Figs. 2B, D, and F). Compared to the H group,
Fig. 2. (A–F) Percent changes of microcirculatory blood flow intensity (2A, 2C, and 2E) and tissue oxygen saturation (2B, 2D, and 2F) at T1, T2, and T3 compared with T0 (2A and 2B: T1 vs. T0; 2C and 2D: T2 vs. T0; and 2E and 2F: T3 vs. T0). Blank bars with bold edges represent changes among the splanchnic organs of the H group. Bars with a grey slash represent changes among the splanchnic organs of the R group. The groups marked with 1, 2, and 3 have differing average percent changes of microcirculatory blood flow intensity (1 b 2 b 3, p b 0.05). The groups marked with ^ represent a significant change between the H and R groups.
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fluid resuscitation did not significantly improve tissue oxygen saturation (Figs. 2B, D, and F). Discussion In this study, we examined a rat model that could 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
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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; and (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
Fig. 3. (A–G) Example of an exposed viscera (Fig. 3A) and laser speckle contrast imaging of the microcirculatory blood flow intensities in the S (3B and 3E), H (3C and 3F), and R (3D and 3G) groups at T0 and T3. The regions of interest were marked as liver (1), kidney (2), intestinal mucosa (3), serosal muscular layer (4), Peyer's patch (5), and gracilis muscle (6).
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time; therefore, it enables simultaneous measurement of changes in microcirculatory blood flow intensity among multiple organs (Beilman et al., 1999; Morini et al., 2000; Sordia et al., 2004; van Iterson et al., 2012). 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 a comparable or even improved reproducibility of microcirculation in comparison with other techniques such as laser Doppler flowmetry or sidestream dark-field (SDF) imaging (Rousseau et al., 2011; Roustit et al., 2010; Sturesson et al., 2013; Tew 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 et al. found that the intestine was more sensitive to hemorrhaging than the heart in a pig model of hemorrhagic shock (Vajda et al., 2004). Additionally, we found that, with heterogeneous impairment of microcirculatory blood flow intensity within the 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 hemodynamics, 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. 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 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) (Jakob, 2002; Rasmussen et al., 1999). 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
Table 3 Splanchnic organ tissue oxygen saturation changes secondary to hemorrhagic shock. StO2 (%) Liver
Kidney
Intestinal mucosa
Serosal muscular layer
Peyer's patch
Gracilis muscle
S H R S H R S H R S H R S H R S H R
T0
T1
T2
T3
ANOVA group
ANOVA time
ANOVA interaction
66.4 ± 2.5 63.9 ± 1.0 57.8 ± 2.4 77.5 ± 1.5 70.1 ± 1.4 71.5 ± 3.0 56.8 ± 1.5 55.8 ± 3.2 57.0 ± 2.0 63.5 ± 2.0 68.2 ± 2.2 66.7 ± 2.8 58.8 ± 1.9 64.8 ± 3.3 65.0 ± 1.3 40.7 ± 3.0 55.2 ± 2.0⁎ 43.2 ± 4.2
69.9 ± 0.6 14.7 ± 3.4⁎# 16.7 ± 5.4⁎# 79.53 ± 1.7 29.2 ± 4.2⁎# 32.8 ± 7.4⁎#
68.03 ± 2.4 38.0 ± 4.0⁎# 39.2 ± 5.6⁎# 76.6 ± 1.7 50.5 ± 2.7⁎# 56.1 ± 2.9⁎#
64.18 ± 2.5 45.8 ± 7.1⁎# 45.8 ± 4.7⁎# 79.8 ± 1.6 54.0 ± 2.9⁎# 56.2 ± 5.5⁎#
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
53.2 ± 2.1 10.1 ± 3.8⁎# 4.3 ± 2.2⁎# 58.6 ± 2.9 18.0 ± 3.5⁎# 11.0 ± 4.7⁎#
49.9 ± 1.9 24.4 ± 4.9⁎# 29.1 ± 3.7⁎# 59.2 ± 0.9 39.2 ± 3.2⁎# 46.0 ± 4.9⁎#
50.5 ± 2.2 34.1 ± 2.7⁎# 31.4 ± 3.8⁎# 59.6 ± 2.1 46.2 ± 4.5⁎# 44.0 ± 4.2⁎#
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
p b 0.001
57.6 ± 1.8 27.8 ± 6.1⁎# 19.2 ± 5.1⁎#
59.0 ± 1.5 46.3 ± 3.6⁎# 54.3 ± 4.1# 39.4 ± 2.1 38.7 ± 2.0# 28.5 ± 1.7#
60.1 ± 1.5 46.0 ± 3.3⁎# 50.9 ± 3.3⁎# 45.2 ± 2.5 45.8 ± 4.6 38.0 ± 2.8
p = 0.008
p b 0.001
p b 0.001
p = 0.016
p b 0.001
p b 0.001
44.4 ± 2.2 31.1 ± 4.6⁎# 21.3 ± 6.2⁎#
S, sham operation group; H, hemorrhagic shock group; StO2, tissue oxygen saturation. ⁎ p b 0.05 compared with sham. # p b 0.05 compared with baseline value.
C.-Y. Wu et al. / Microvascular Research 101 (2015) 55–61
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 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 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. We suggest that this model be applied in future studies investigating the effects of different methods of resuscitation for hemorrhagic shock on microcirculatory changes among splanchnic organs. Acknowledgments This study was supported in part by the research grant NTUH.103S2361 from National Taiwan University Hospital. We thank Zong-Gin Wu (Technician, Department of Surgery, National Taiwan University Hospital) and Roger Lien (Technician, MicroStar Instruments Co., Ltd., Taipei, Taiwan) for their technical assistance.
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