Resuscitation 85 (2014) 833–839
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Experimental paper
Post-resuscitation intestinal microcirculation: Its relationship with sublingual microcirculation and the severity of post-resuscitation syndrome夽,夽夽 Jie Qian a,c , Zhengfei Yang a , Jena Cahoon a , Jiefeng Xu a , Changqing Zhu c,∗ , Min Yang a , Xianwen Hu a , Shijie Sun a,b , Wanchun Tang a,b,∗∗ a
Weil Institute of Critical Care Medicine, Rancho Mirage, CA, United States Keck School of Medicine of the University of Southern California, Los Angeles, CA, United States c Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 10 January 2014 Accepted 20 February 2014 Keywords: Post-resuscitation syndrome Microcirculation Sublingual Intestine Inflammatory response
a b s t r a c t Objective: Post-resuscitation syndrome has been recognized as one of the major causes of the poor outcomes of cardiopulmonary resuscitation. The aims of this study were to investigate the intestinal microcirculatory changes following cardiopulmonary resuscitation and relate those changes to sublingual microcirculation and the severity of post-resuscitation syndrome as measured by myocardial function and serum inflammatory cytokine levels. Methods: Twenty-five rats were randomized into three groups: (1) short duration of cardiac arrest (n = 10): ventricular fibrillation (VF) was untreated for 4 min prior to 6 min of cardiopulmonary resuscitation (CPR); (2) long duration of cardiac arrest (n = 10): VF was untreated for 8 min followed by 8 min of CPR; (3) sham control group (n = 5): a sham operation was performed without VF induction and CPR. Intestinal and sublingual microcirculatory blood flow was visualized by a sidestream dark-field (SDF) imaging device at baseline and 1, 2, 4, 6, 8 h post-resuscitation. Myocardial function was measured by echocardiography and serum cytokine levels (TNF-␣ and IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). Results: Both intestinal and sublingual microcirculatory blood flow decreased significantly with increasing duration of cardiac arrest and resuscitation. The decreases in intestinal microcirculatory blood flow were closely correlated with the reductions of sublingual microcirculatory blood flow (perfused small vessels density: r = 0.772, p < 0.01; microcirculatory flow index: r = 0.821, p < 0.01). The decreased microcirculatory blood flow was closely correlated with weakened myocardial function and elevated inflammatory cytokine levels. Conclusions: The severity of post-resuscitation intestinal microcirculatory dysfunction is closely correlated with that of myocardial function and inflammatory cytokine levels. The measurement of sublingual microcirculation reflects changes of intestinal microcirculation and may therefore provide a new option for post-resuscitation monitoring. © 2014 Elsevier Ireland Ltd. All rights reserved.
夽 A Spanish translated version of the abstract of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2014.02.019. 夽夽 Protocol number: R1304. ∗ Corresponding author at: Department of Emergency Medicine, Renji Hospital, Shanghai JiaoTong University School of Medicine, 1630# Dongfang Road, Pudong New District, Shanghai 200127, China. ∗∗ Corresponding author at: Weil Institute of Critical Care Medicine, 35100 Bob Hope Drive, Rancho Mirage, CA 92270, United States. E-mail addresses: [email protected] (J. Qian), [email protected] (Z. Yang), [email protected] (J. Cahoon), [email protected] (J. Xu), [email protected] (C. Zhu), [email protected] (M. Yang), [email protected] (X. Hu), [email protected] (S. Sun), [email protected] (W. Tang). http://dx.doi.org/10.1016/j.resuscitation.2014.02.019 0300-9572/© 2014 Elsevier Ireland Ltd. All rights reserved.
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1. Introduction Though approximately 50% of cardiac arrest (CA) victims are successfully resuscitated initially, only 5–15% survive to hospital discharge.1,2 The prognosis after CA and resuscitation remains poor. Post-resuscitation syndrome is a complex state characterized by myocardial dysfunction, brain injury, global ischemia–reperfusion (I/R) injury and systemic inflammatory response. It accounts partly for the poor outcome.3 The intestine is likely to be the most sensitive to I/R injury among the internal organs. A previous study demonstrated that CA caused a prolonged reduction of blood flow to the intestine.4 As a result, the intestinal permeability increases and a systemic inflammatory response may subsequently be triggered.5 This has been considered an important mechanism of sepsis.6 The elevated inflammatory cytokines after cardiopulmonary resuscitation mimics that of sepsis.7 However, whether the intestinal circulatory dysfunction contributes to the severity of post-resuscitation syndrome remains unclear. Current evidence indicated that microcirculation regulates tissue blood flow and is therefore more important than macrocirculation on tissue oxygen delivery and utilization.8,9 Microcirculatory dysfunction has been recognized as an important determinant of the outcome in circulatory shock.10,11 Microcirculatory changes differ extensively among organs especially during the low-flow states.4,9,12–14 The sublingual area is easy to access and has been considered an ideal location to evaluate microcirculation. However, whether it fully reflects visceral microcirculation remains unclear. In the present study, we investigated the concurrent changes of intestinal and sublingual microcirculation, cardiac function and inflammatory cytokine levels in a rat model of cardiopulmonary resuscitation (CPR). Our hypotheses were that: (1) Intestinal microcirculatory dysfunction is closely correlated with the severity of post-resuscitation syndrome as measured by myocardial function and inflammatory cytokine levels. (2) The changes of sublingual microcirculation during post-resuscitation reflect changes of intestinal microcirculation. 2. Materials and methods All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (8th edition, Washington, DC, National Academies Press, 2011). The protocol was approved by the Institutional Animal Care and Use Committee of the Weil Institute of Critical Care Medicine. 2.1. Animal preparation Twenty-five male Sprague–Dawley rats weighing 450–550 g were fasted overnight except for free access to water. The animals were anesthetized by intraperitoneal injection of pentobarbital (45 mg/kg). Additional doses (10 mg/kg) were administered at hourly intervals. The trachea was orally intubated with a 14-gauge cannula (Abbocath-T; Abbott Hospital, North Chicago, IL). The animals were breathing room-air spontaneously. End-tidal CO2 (ETCO2 ) was continuously monitored with a sidestream infrared CO2 analyzer (model 200; Instrumentation Laboratories, Lexington, MA). Two 23-gauge PE-50 catheters (Becton-Dickinson, Franklin Lakes, NJ) were advanced through the left external jugular vein into the right atrium and through the left femoral artery into the descending aorta for measurement of right atrial pressure and aortic pressure with high-sensitivity transducers (model 42584-01;
Abbott Critical Care Systems, North Chicago, IL). A thermocouple microprobe, 10 cm in length and 0.5 mm in diameter (9030-12D-34; Columbus Instruments, Columbus, OH), was inserted into the left femoral vein to measure blood temperature. A 3F PE catheter (model C-PMS-301J; Cook Critical Care, Bloomington, IN) was advanced through the right external jugular vein into the right atrium. All catheters were flushed intermittently with saline containing 2.5 IU/mL crystalline bovine heparin. A conventional lead II ECG was continuously monitored. The blood temperature for all animals was maintained at 37 ◦ C ± 0.5 ◦ C with a heating lamp. A laparotomy was performed to expose the peritoneal cavity through the midline abdominal incision (∼1.5 cm). The wound was covered by sterile 37 ◦ C warmed normal saline-saturated gauze to minimize dehydration and loss of body heat. 2.2. Experimental procedures The animals were randomly assigned to one of three groups: (1) short duration of CA (n = 10): ventricular fibrillation (VF) was untreated for 4 min prior to 6 min of cardiopulmonary resuscitation (CPR); (2) long duration of CA (n = 10): VF was untreated for 8 min followed by 8 min of CPR; (3) sham control group (n = 5): a sham operation was performed without VF induction and CPR. Ten minutes prior to inducing VF, baseline measurements were obtained and mechanical ventilation was initiated at a tidal volume of 0.60 ml/100 g body weight, a frequency of 100 breaths/min and an inspired O2 fraction (FiO2 ) of 0.21. VF was electrically induced with a progressive increase in 60-Hz current to a maximum of 3.5 mA delivered to the right ventricular endocardium. The current flow was continued for 3 min to prevent spontaneous defibrillation. Mechanical ventilation was discontinued after onset of VF. Precordial compression (PC) was started after either 4 or 8 min of VF. Coincident with the start of PC, the animals were mechanically ventilated at a frequency of 100 breaths per minute and with an FiO2 of 1.0. PC was maintained at a rate of 200/min and synchronized to provide a compression/ventilation ratio of 2:1 with equal compression–relaxation. The depth of compressions was initially adjusted to maintain a coronary perfusion pressure (CPP: diastolic aortic pressure minus right atrial pressure) at 22 ± 2 mmHg with ETCO2 11 ± 2 mmHg. Resuscitation was attempted with up to 3 two-joule countershocks. If return of spontaneous circulation (ROSC) was not achieved, a 30-s interval of CPR was performed before a subsequent sequence of up to 3 shocks was attempted. This procedure was repeated for a maximum of 3 cycles. ROSC was defined as the return of supraventricular rhythm with a mean aortic pressure above 50 mmHg for a minimum of 5 min. Following ROSC, mechanical ventilation was continued with 100% oxygen for the first hour, 50% for the second hour and 21% thereafter. There was no pharmacology agent used during CPR. After the experiment, all animals were euthanized by an overdose of pentobarbital (150 mg/kg via the femoral artery). Abdominal cavity culture was performed to prove the laparotomy was sterile. A necropsy was performed to document injuries to thoracic, abdominal vessels and viscera caused by surgical intervention. 2.3. Measurements Aortic and right atrial pressures, electrocardiogram and ETCO2 were continuously recorded for 8 h on a personal computer-based data acquisition system (DATAQ Instruments, Akron, OH). Myocardial function, including cardiac output (CO), ejection fraction (EF) and myocardial performance index (MPI), were
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assessed at baseline, 1, 2, 4, 6 and 8 h after ROSC using echocardiography (HD 11 XE, Philips Healthcare, Andover, MA) with a 12.5 MHz transducer.15 Artery blood samples (0.3 ml) were withdrawn at baseline, 2, 4, 6 and 8 h after ROSC for measuring arterial blood gases with the aid of a Stat Profile pHOx analyzer (Nova Biomedical Corporation, Waltham, MA). For the measurement of serum cytokine, blood samples (1.2 ml) were collected at the same time points and immediately centrifuged at 3000 rpm for 10 min. Then, the serum was stored until analysis. The experimental rat received an equivalent volume (1.5 ml) of arterial blood from a donor rat after the blood withdrawal. TNF-␣ and IL-6 concentrations were determined by using ELISA kits (TNF-␣: R&D Systems, Minneapolis, MN; IL-6: Thermo Scientific, Rockford, IL). The experiments were performed following the manufacturer’s instructions. Microcirculations were visualized at baseline, 1, 2, 4, 6 and 8 h after ROSC with the aid of a sidestream dark-field (SDF) imaging device (MicroScan; MicroVision Medical Inc., Amsterdam, Netherlands) with a 5× optical probe. The sublingual region was assessed near the base of the tongue. For the measurements of intestinal microcirculation, a 2–3 cm segment of the jejunum was withdrawn with its neurovascular supply intact and cushioned with warm saline-soaked gauze. The jejunal microcirculation was assessed on the anti-mesenteric aspect of the serosal side. After microcirculatory measurements were taken, the abdominal contents were then returned into the peritoneal cavity and the abdomen was closed in two layers. The muscular layer of the abdominal wall was sutured in a continuous pattern, and the skin incision was closed with wound clips. Three discrete fields were captured with precaution to minimize motion and pressure artifacts. Microvascular images were recorded on a DVD disk using a DVD recorder (Model DMREZ47V; Panasonic AVC Networks, Dalian, China). Individual images were analyzed offline. Microcirculatory flow index (MFI) was quantitated by the method of Boerma et al.16 The image was divided into four quadrants and the predominant type of flow (absent = 0, intermittent = 1, sluggish = 2 and normal = 3) was assessed in the small vessels of each quadrant, which were less than 20 m in diameter. The MFI score represented the average values of four quadrants. Perfused vessel density (PVD) was measured based on the method of De Backer et al.17 Vessel density was calculated as the number of vessels crossing the lines divided by the total length of the lines. Vessel size was measured with a micrometer scale superimposed in the video display. All recordings were analyzed by three independent observers. 2.4. Statistical analyses Measurements were reported as mean ± SD or median (interquartile range) as indicated. Variables were compared with one-way ANOVA or the Kruskal–Wallis test for nonparametric data. Comparisons between time-based measurements within each group were performed with repeated-measurement analysis of variance. If there was a significant difference in the overall comparison of groups, comparisons between any other two groups were made by the Bonferroni test. Linear correlations were calculated using the Pearson correlation coefficient. A value of p < 0.05 was considered significant. 3. Results Baseline hemodynamics, myocardial function and blood analytical measurements did not differ among groups (Table 1). All animals were successfully resuscitated. There was no difference in the amount of the electric current required to induce VF between CPR groups. Body temperature did not differ significantly
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Table 1 Baseline characteristics. Group
Sham control
Body weight, g Heart rate, bpm MAP, mmHg End-tidal CO2 , mmHg Ejection fraction, % Cardiac output, mL/min MPI pH Lactate, mmol/L
510 346 135 41 70.0 107.0 0.71 7.48 0.9
± ± ± ± ± ± ± ± ±
24 21 6 1 1.0 5.2 0.04 0.03 0.3
Short duration of CA 506 356 133 41 69.5 107.3 0.69 7.51 0.8
± ± ± ± ± ± ± ± ±
15 28 10 1 1.5 2.6 0.04 0.03 0.1
Long duration of CA 503 357 136 41 69.5 107.6 0.68 7.50 0.9
± ± ± ± ± ± ± ± ±
14 24 8 1 1.7 4.1 0.03 0.04 0.2
CA, cardiac arrest; MAP, mean aortic pressure; MPI, myocardial performance index. Values are presented as mean ± SD.
among groups during the experiment. There was no significant difference of CPP between the CPR groups during CPR. CPP at the end of CPR was 25.0 ± 3.2 mmHg in the short duration group and was 24.5 ± 2.9 mmHg in the long duration group. The number of defibrillations did not differ significantly (1.4 ± 0.5 vs. 2.1 ± 2.2) between the short and the long duration groups. The post-resuscitation PVD decreased in parallel with the reduced MFI in both intestinal and sublingual sites. In the long duration group, intestinal PVD was significantly reduced from baseline of 19.8 ± 1.7 to 10.1 ± 1.8 n/mm at 8 h post-resuscitation and MFI from 3.0 ± 0.0 to 1.4 ± 0.7 (both p < 0.05 vs. baseline and sham control). Similarly, sublingual PVD was significantly reduced from the baseline of 5.6 ± 0.8 to 2.3 ± 0.6 n/mm at 8 h post-resuscitation and MFI from 3.0 ± 0.0 to 1.3 ± 0.6 (both p < 0.05 vs. baseline and sham control). Contrary to a progressive recovery of both measurements observed in the short duration group, the significant reduction in both PVD and MFI persisted after resuscitation in the long duration group. At 8 h post-resuscitation, PVD and MFI were significantly lower in the long duration group than in the short duration group: intestinal PVD (10.1 ± 1.8 vs. 17.7 ± 2.8 n/mm, p < 0.05); sublingual PVD (2.3 ± 0.6 vs. 4.8 ± 0.9 n/mm, p < 0.05); intestinal MFI (1.4 ± 0.7 vs. 2.4 ± 0.4, p < 0.05); sublingual MFI (1.3 ± 0.6 vs. 2.4 ± 0.5, p < 0.05). The decreases in intestinal microcirculatory blood flow were closely correlated with the reductions of sublingual microcirculatory blood flow (PVD: r = 0.772, p < 0.01; MFI: r = 0.821, p < 0.01) (Fig. 1). Examples of the images of intestinal and sublingual microvasculature, as obtained by the SDF video microscope at 8 h post-resuscitation, are shown in Fig. 2. Myocardial function was significantly impaired in the postresuscitation period in both CPR groups compared with baseline values. Significantly worse post-resuscitation myocardial function was observed after long duration of CA when compared to the short duration of CA group which gradually recovered over time (Fig. 3). Serum cytokine levels in both CPR groups elevated after resuscitation. There was significantly elevated TNF-␣ levels in the long duration group 2 h after resuscitation. In the short duration group, there was a significant elevation only 8 h after resuscitation. However, for IL-6, except in the long duration group at 6 and 8 h post-resuscitation, none were statistically different from the sham control group (Fig. 4). There were significant correlations between MFI, PVD, myocardial function and serum cytokine levels (Table 2). At 8 h post-resuscitation, ETCO2 values in the long duration group were significantly lower than those of the sham control and the short duration group. Plasma lactate levels in the long duration group remained significantly greater when compared with the sham control and the short duration group (p < 0.05). However, there was no significant difference in heart rate or mean aortic pressure (MAP) between the CPR groups (Table 3).
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Fig. 1. Changes of microcirculatory variables and the correlation between sublingual and intestinal microcirculation. BL, baseline; CA, cardiac arrest; MFI, microcirculatory flow index; PVD, perfused vessel density. *p < 0.05, vs. sham control group. # p < 0.05 vs. short duration of CA group.
4. Discussion The present study demonstrated that both intestinal and sublingual microcirculatory blood flow decreased significantly with increasing duration of cardiac arrest and resuscitation. Intestinal microcirculatory dysfunction is closely correlated with the severity of post-resuscitation syndrome as measured by myocardial Table 2 Correlational analysis between myocardial function, microcirculatory parameters and serum cytokine levels. Sublingual
Intestinal
PVD
MFI
PVD
MFI
Cardiac function CO EF MPI
0.827* 0.846* −0.794*
0.846* 0.823* −0.775*
0.821* 0.767* −0.750*
0.839* 0.818* −0.757*
Cytokine levels TNF-␣ IL-6
−0.332* −0.415*
−0.380* −0.394*
−0.420* −0.406*
−0.307* −0.326*
PVD, perfused vessel density; MFI, microcirculatory flow index; CO, cardiac output; EF, ejection fraction; MPI, myocardial performance index; TNF-␣, tumor necrosis factor-alpha; IL-6, interleukin-6. * p < 0.01
function and inflammatory cytokine levels. Sublingual microcirculatory changes were closely correlated with that of intestinal. In humans, the easiest accessible site to detect microcirculation is the sublingual area. Sublingual microcirculation is considered as a surrogate measure for splanchnic blood flow. Under conditions of circulatory shock, noninvasive sublingual capnometry yielded measurements that were interchangeable with those of gastric tonometry.18–20 Using a direct visualization approach, our findings demonstrated the similar behavior of sublingual and intestinal microcirculation at the early stage after ROSC. Our findings are
Table 3 Characteristics at 8 h post-resuscitation. Group
Sham control
Heart rate, bpm MAP, mmHg End-tidal CO2 , mmHg pH Lactate, mmol/L
337 126 40 7.54 1.1
± ± ± ± ±
27 4 1 0.02 0.2
Short duration of CA 319 104 36 7.54 1.3
± ± ± ± ±
51 13* 2* 0.04 0.4
Long duration of CA 342 97 29 7.51 2.4
± ± ± ± ±
28 17* 6* , # 0.09 0.9* , #
CA, cardiac arrest; MAP indicates mean aortic pressure; Values are presented as mean ± SD. * p < 0.05 vs. sham control group. # p < 0.05 vs. short duration of CA group.
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Fig. 2. Images of sublingual and intestinal microcirculation obtained by the sidestream dark-field (SDF) video microscope at 8 h post-resuscitation.
consistent with earlier studies not only of circulatory shock but also of endotoxic and septic shock, which indicates that the severity and the time course of microcirculatory changes were similar in the sublingual and gut region.21–24 Interestingly, two studies on sepsis demonstrated conflicting findings on the relationship between these two sites. Though both revealed time-varying correlations between these two sites, a clinical study claimed that microcirculatory changes were not parallel on day 1 while another animal study demonstrated that the initial strong correlation disappeared over time.25,26 Highly heterogeneous nature of the sepsis victims might explain the conflicting results. Additionally, both studies applied therapeutic interventions that may further influence the intestinal microcirculation.27,28 Although post-resuscitation abnormalities mimic the disorders observed in sepsis, cardiac dysfunction is one of the primary distinguishing characteristics. The impairment of cardiac function during the post-resuscitation period can precipitate a downward splanchnic blood flow. However, microcirculatory changes can be dispatched from changes in regional blood flow and differ among visceral organs especially in low-flow states.29 In the present study, we revealed a close correlation between cardiac function and microcirculatory changes in sublingual and intestinal sites in the early stage following resuscitation. Noticeably, in the long duration of the CA group, cardiac function deteriorated over time, intestinal microcirculatory parameters experienced a slightly upward trend after PR 1 h and then dropped to approximately 50% of baseline value at PR 8 h. The autoregulation may account for the fluctuation of microcirculatory blood flow. In fact, the regulation of microcirculatory blood flow in the intestine wall is extremely complicated. Not only systemic factors but also local factors such as inflammatory cytokines, vasomotor function and metabolic products affect the intestinal microcirculation. Under this experimental condition, we speculate that the intestinal microcirculatory dysfunction caused by systemic insults was beyond the limit of the autoregulatory threshold.
The severity of intestinal microcirculatory dysfunction was magnified when the duration of cardiac arrest and resuscitation was prolonged. In the present study, intestinal microcirculation was impaired in both CPR groups. A partially reversible injury within 8 h was observed in the short duration group. This pattern of response was consistent with previous studies that demonstrated a transition from reversible to irreversible intestinal damage as duration of ischemia prolonged.30 In Korth et al.’s study, 4 min of cardiac arrest caused a resembled reduction of intestinal blood flow during the following 60 min. Though they did not observe blood flow thereafter, this phenomenon that the intestinal metabolic changes were gradually restored over time may indicate a resembled recovery of intestinal microcirculation.4 There may be several factors contributing to the fact that prolonged cardiac arrest leads to more severe intestinal microcirculatory derangement. First, as mentioned above, prolonged CA results in more pronounced myocardial dysfunction, the intestinal microcirculation is therefore more likely to be injured as a result of reduced cardiac output. Second, within 4–6 min of CA, high-energy phosphates become depleted, thus favor disruption of microvascular endothelial cells.31 Third, longer duration of CA may result in local intestinal thrombosis and leukocyte plugging, thus causes the decrease in capillary flow.32 Intestinal barrier integrity plays a pivotal role in the development of systemic inflammatory response syndrome, sepsis and multiple organ failure. The intestine is not only a vulnerable target to ischemia but a possible secondary source of inflammatory cytokines as well. We cannot rule out the possibility that postresuscitation disturbed intestinal microcirculation may trigger an inflammatory cascade. IL-6 as well as TNF-␣ are key mediators of the systemic inflammatory response. Our results confirmed an intense increase of such cytokines in the early hours after CA.7 The decreases of microcirculatory blood flow were correlated with the increases of inflammatory cytokine levels. In our study, a discrepant time course of TNF-␣ and IL-6 levels after different durations of CA
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Fig. 4. Serum TNF-␣ and IL-6 concentrations at baseline and post-resuscitation (mean ± SD). *p < 0.05, vs. sham control group. # p < 0.05 vs. short duration of CA group.
Fig. 3. Myocardial function at baseline and post-resuscitation. EF, ejection fraction; CO, cardiac output; MPI, myocardial performance index; BL, baseline; CA, cardiac arrest; VF, ventricular fibrillation. Values are presented as mean ± SD. *p < 0.05, vs. sham control group. # p < 0.05 vs. short duration of CA group.
were observed. The TNF-␣ level remained at high values 2 h after ROSC in the long duration of the CA group while there was a 6 h lag after short duration. IL-6 expression, which can be induced by TNF␣, elevated at 6 h post-resuscitation after 8 min of CA. Although the present study was not designed to compare survival rates among groups, several studies have confirmed high levels of TNF-␣ and IL-6 could discriminate between survivors and nonsurvivors.7,33 Clinically, intestine injuries after cardiac arrest are often underestimated though it might be a potentially devastating complication even following successful resuscitation.34 The manifestations may be nonspecific and delayed. The direct in vivo observation of the intestinal microcirculation is not clinically feasible and making early detections is therefore difficult. Our study demonstrated that the changes of sublingual microcirculation during post-resuscitation reflected changes of intestinal microcirculation, myocardial function and inflammatory cytokine levels. This suggests that sublingual microcirculation may therefore provide a new option to monitor post-resuscitation patients at the bedside. To interpret the results of our experimental study, it is necessary to take several limitations into consideration. First, therapeutic intervention was not applied in the study, which is different from
clinical situations. Microcirculation can be affected by several factors. The use of epinephrine, hypothermia or fluid therapy may have reduced differences in microcirculation parameters between groups. Second, we investigated the microvasculature of serosa layer rather than the mucosa layer. It is likely that microcirculatory changes in these two sites are different. Third, we didn’t investigate the neurologic function which is one important component of post-resuscitation syndrome. Fourth, using SDF imaging, PVD and MFI were evaluated in a semiquantitative way. A quantitative analysis might generate a more accurate outcome. Fifth, movement and pressure artifacts hinder the analysis of the images though we tried to reduce the interference. Finally, we did not measure the abdominal pressures which might affect the microcirculatory blood flow in the intestinal wall.35 However, a sham control group was enrolled to rule out this factor. 5. Conclusion The severity of post-resuscitation intestinal microcirculatory dysfunction is closely correlated with that of myocardial function and inflammatory cytokine levels. The measurement of sublingual microcirculation reflects changes of intestinal microcirculation and may therefore provide a new option for post-resuscitation monitoring. Conflict of interest None of the authors have any conflict of interest to report. Acknowledgement Lisa Luna contributed to the editing of this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.resuscitation. 2014.02.019.
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17. De Backer D, Creteur J, Preiser JC, Dubois MJ, Vincent JL. Microvascular blood flow is altered in patients with sepsis. Am J Respir Crit Care Med 2002;166:98–104. 18. Povoas HPWM, Tang W, Moran B, Kamohara T, Bisera J. Comparisons between sublingual and gastric tonometry during hemorrhagic shock. Chest 2000;118:1127–32. 19. Weil MH, Nakagawa Y, Tang W, et al. Sublingual capnometry: a new noninvasive measurement for diagnosis and quantitation of severity of circulatory shock. Crit Care Med 1999;27:1225–9. 20. Creteur J, De Backer D, Sakr Y, Koch M, Vincent JL. Sublingual capnometry tracks microcirculatory changes in septic patients. Intensive Care Med 2006;32:516–23. 21. Verdant CL, De Backer D, Bruhn A, et al. Evaluation of sublingual and gut mucosal microcirculation in sepsis: a quantitative analysis. Crit Care Med 2009;37:2875–81. 22. Guzman JA, Dikin MS, Kruse JA. Lingual, splanchnic, and systemic hemodynamic and carbon dioxide tension changes during endotoxic shock and resuscitation. J Appl Physiol 2005;98:108–13. 23. Dubin A, Edul VS, Pozo MO, et al. Persistent villi hypoperfusion explains intramucosal acidosis in sheep endotoxemia. Crit Care Med 2008;36:535–42. 24. Fries M, Weil MH, Sun S, et al. Increases in tissue pco2 during circulatory shock reflect selective decreases in capillary blood flow. Crit Care Med 2006;34:446–52. 25. Boerma EC, vdVP, Spronk PE, Ince C. Relationship between sublingual and intestinal microcirculatory perfusion in patients with abdominal sepsis. Crit Care Med 2007;35:1055–60. 26. Pranskunas A, Pilvinis V, Dambrauskas Z, et al. Early course of microcirculatory perfusion in eye and digestive tract during hypodynamic sepsis. Crit Care 2012;16:R83. 27. Oktar BK, Gulpinar MA, Bozkurt A, et al. Endothelin receptor blockers reduce i/rinduced intestinal mucosal injury: role of blood flow. Am J Physiol Gastrointest Liver Physiol 2002;282:G647–55. 28. Nacul FE, Guia IL, Lessa MA, Tibirica E. The effects of vasoactive drugs on intestinal functional capillary density in endotoxemic rats: intravital videomicroscopy analysis. Anesth Analg 2010;110:547–54. 29. Hiltebrand LB, Krejci V, Banic A, Erni D, Wheatley AM, Sigurdsson GH. Dynamic study of the distribution of microcirculatory blood flow in multiple splanchnic organs in septic shock. Crit Care Med 2000;28:3233–41. 30. Guan Y, Worrell RT, Pritts TA, Montrose MH. Intestinal ischemia–reperfusion injury: reversible and irreversible damage imaged in vivo. Am J Physiol Gastrointest Liver Physiol 2009;297:G187–96. 31. Padosch SA, Teschendorf P, Fuchs A, et al. Effects of abciximab on postresuscitation microcirculatory dysfunction after experimental cardiac arrest in rats. Resuscitation 2010;81:255–9. 32. Schoots IG, Levi M, Roossink EH, Bijlsma PB, van Gulik TM. Local intravascular coagulation and fibrin deposition on intestinal ischemia–reperfusion in rats. Surgery 2003;133:411–9. 33. Michael A, Basha G, Scott M, Steven LK, Robert MS, Emanuel PR, et al. Presence of tumor necrosis factor in humans undergoing cardiopulmonary resuscitation with return of spontaneous circulation. J Crit Care 1991;6:185–9. 34. Katsoulis IE, Balanika A, Sakalidou M, Gogoulou I, Stathoulopoulos A, Digalakis MK. Extensive colonic necrosis following cardiac arrest and successful cardiopulmonary resuscitation: report of a case and literature review. World J Emerg Surg 2012;7:35. 35. Samel ST, Neufang T, Mueller A, Leister I, Becker H, Post S. A new abdominal cavity chamber to study the impact of increased intra-abdominal pressure on microcirculation of gut mucosa by using video microscopy in rats. Crit Care Med 2002;30:1854–8.
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Experimental paper
Post-resuscitation intestinal microcirculation: Its relationship with sublingual microcirculation and the severity of post-resuscitation syndrome夽,夽夽 Jie Qian a,c , Zhengfei Yang a , Jena Cahoon a , Jiefeng Xu a , Changqing Zhu c,∗ , Min Yang a , Xianwen Hu a , Shijie Sun a,b , Wanchun Tang a,b,∗∗ a
Weil Institute of Critical Care Medicine, Rancho Mirage, CA, United States Keck School of Medicine of the University of Southern California, Los Angeles, CA, United States c Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 10 January 2014 Accepted 20 February 2014 Keywords: Post-resuscitation syndrome Microcirculation Sublingual Intestine Inflammatory response
a b s t r a c t Objective: Post-resuscitation syndrome has been recognized as one of the major causes of the poor outcomes of cardiopulmonary resuscitation. The aims of this study were to investigate the intestinal microcirculatory changes following cardiopulmonary resuscitation and relate those changes to sublingual microcirculation and the severity of post-resuscitation syndrome as measured by myocardial function and serum inflammatory cytokine levels. Methods: Twenty-five rats were randomized into three groups: (1) short duration of cardiac arrest (n = 10): ventricular fibrillation (VF) was untreated for 4 min prior to 6 min of cardiopulmonary resuscitation (CPR); (2) long duration of cardiac arrest (n = 10): VF was untreated for 8 min followed by 8 min of CPR; (3) sham control group (n = 5): a sham operation was performed without VF induction and CPR. Intestinal and sublingual microcirculatory blood flow was visualized by a sidestream dark-field (SDF) imaging device at baseline and 1, 2, 4, 6, 8 h post-resuscitation. Myocardial function was measured by echocardiography and serum cytokine levels (TNF-␣ and IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). Results: Both intestinal and sublingual microcirculatory blood flow decreased significantly with increasing duration of cardiac arrest and resuscitation. The decreases in intestinal microcirculatory blood flow were closely correlated with the reductions of sublingual microcirculatory blood flow (perfused small vessels density: r = 0.772, p < 0.01; microcirculatory flow index: r = 0.821, p < 0.01). The decreased microcirculatory blood flow was closely correlated with weakened myocardial function and elevated inflammatory cytokine levels. Conclusions: The severity of post-resuscitation intestinal microcirculatory dysfunction is closely correlated with that of myocardial function and inflammatory cytokine levels. The measurement of sublingual microcirculation reflects changes of intestinal microcirculation and may therefore provide a new option for post-resuscitation monitoring. © 2014 Elsevier Ireland Ltd. All rights reserved.
夽 A Spanish translated version of the abstract of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2014.02.019. 夽夽 Protocol number: R1304. ∗ Corresponding author at: Department of Emergency Medicine, Renji Hospital, Shanghai JiaoTong University School of Medicine, 1630# Dongfang Road, Pudong New District, Shanghai 200127, China. ∗∗ Corresponding author at: Weil Institute of Critical Care Medicine, 35100 Bob Hope Drive, Rancho Mirage, CA 92270, United States. E-mail addresses: [email protected] (J. Qian), [email protected] (Z. Yang), [email protected] (J. Cahoon), [email protected] (J. Xu), [email protected] (C. Zhu), [email protected] (M. Yang), [email protected] (X. Hu), [email protected] (S. Sun), [email protected] (W. Tang). http://dx.doi.org/10.1016/j.resuscitation.2014.02.019 0300-9572/© 2014 Elsevier Ireland Ltd. All rights reserved.
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1. Introduction Though approximately 50% of cardiac arrest (CA) victims are successfully resuscitated initially, only 5–15% survive to hospital discharge.1,2 The prognosis after CA and resuscitation remains poor. Post-resuscitation syndrome is a complex state characterized by myocardial dysfunction, brain injury, global ischemia–reperfusion (I/R) injury and systemic inflammatory response. It accounts partly for the poor outcome.3 The intestine is likely to be the most sensitive to I/R injury among the internal organs. A previous study demonstrated that CA caused a prolonged reduction of blood flow to the intestine.4 As a result, the intestinal permeability increases and a systemic inflammatory response may subsequently be triggered.5 This has been considered an important mechanism of sepsis.6 The elevated inflammatory cytokines after cardiopulmonary resuscitation mimics that of sepsis.7 However, whether the intestinal circulatory dysfunction contributes to the severity of post-resuscitation syndrome remains unclear. Current evidence indicated that microcirculation regulates tissue blood flow and is therefore more important than macrocirculation on tissue oxygen delivery and utilization.8,9 Microcirculatory dysfunction has been recognized as an important determinant of the outcome in circulatory shock.10,11 Microcirculatory changes differ extensively among organs especially during the low-flow states.4,9,12–14 The sublingual area is easy to access and has been considered an ideal location to evaluate microcirculation. However, whether it fully reflects visceral microcirculation remains unclear. In the present study, we investigated the concurrent changes of intestinal and sublingual microcirculation, cardiac function and inflammatory cytokine levels in a rat model of cardiopulmonary resuscitation (CPR). Our hypotheses were that: (1) Intestinal microcirculatory dysfunction is closely correlated with the severity of post-resuscitation syndrome as measured by myocardial function and inflammatory cytokine levels. (2) The changes of sublingual microcirculation during post-resuscitation reflect changes of intestinal microcirculation. 2. Materials and methods All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (8th edition, Washington, DC, National Academies Press, 2011). The protocol was approved by the Institutional Animal Care and Use Committee of the Weil Institute of Critical Care Medicine. 2.1. Animal preparation Twenty-five male Sprague–Dawley rats weighing 450–550 g were fasted overnight except for free access to water. The animals were anesthetized by intraperitoneal injection of pentobarbital (45 mg/kg). Additional doses (10 mg/kg) were administered at hourly intervals. The trachea was orally intubated with a 14-gauge cannula (Abbocath-T; Abbott Hospital, North Chicago, IL). The animals were breathing room-air spontaneously. End-tidal CO2 (ETCO2 ) was continuously monitored with a sidestream infrared CO2 analyzer (model 200; Instrumentation Laboratories, Lexington, MA). Two 23-gauge PE-50 catheters (Becton-Dickinson, Franklin Lakes, NJ) were advanced through the left external jugular vein into the right atrium and through the left femoral artery into the descending aorta for measurement of right atrial pressure and aortic pressure with high-sensitivity transducers (model 42584-01;
Abbott Critical Care Systems, North Chicago, IL). A thermocouple microprobe, 10 cm in length and 0.5 mm in diameter (9030-12D-34; Columbus Instruments, Columbus, OH), was inserted into the left femoral vein to measure blood temperature. A 3F PE catheter (model C-PMS-301J; Cook Critical Care, Bloomington, IN) was advanced through the right external jugular vein into the right atrium. All catheters were flushed intermittently with saline containing 2.5 IU/mL crystalline bovine heparin. A conventional lead II ECG was continuously monitored. The blood temperature for all animals was maintained at 37 ◦ C ± 0.5 ◦ C with a heating lamp. A laparotomy was performed to expose the peritoneal cavity through the midline abdominal incision (∼1.5 cm). The wound was covered by sterile 37 ◦ C warmed normal saline-saturated gauze to minimize dehydration and loss of body heat. 2.2. Experimental procedures The animals were randomly assigned to one of three groups: (1) short duration of CA (n = 10): ventricular fibrillation (VF) was untreated for 4 min prior to 6 min of cardiopulmonary resuscitation (CPR); (2) long duration of CA (n = 10): VF was untreated for 8 min followed by 8 min of CPR; (3) sham control group (n = 5): a sham operation was performed without VF induction and CPR. Ten minutes prior to inducing VF, baseline measurements were obtained and mechanical ventilation was initiated at a tidal volume of 0.60 ml/100 g body weight, a frequency of 100 breaths/min and an inspired O2 fraction (FiO2 ) of 0.21. VF was electrically induced with a progressive increase in 60-Hz current to a maximum of 3.5 mA delivered to the right ventricular endocardium. The current flow was continued for 3 min to prevent spontaneous defibrillation. Mechanical ventilation was discontinued after onset of VF. Precordial compression (PC) was started after either 4 or 8 min of VF. Coincident with the start of PC, the animals were mechanically ventilated at a frequency of 100 breaths per minute and with an FiO2 of 1.0. PC was maintained at a rate of 200/min and synchronized to provide a compression/ventilation ratio of 2:1 with equal compression–relaxation. The depth of compressions was initially adjusted to maintain a coronary perfusion pressure (CPP: diastolic aortic pressure minus right atrial pressure) at 22 ± 2 mmHg with ETCO2 11 ± 2 mmHg. Resuscitation was attempted with up to 3 two-joule countershocks. If return of spontaneous circulation (ROSC) was not achieved, a 30-s interval of CPR was performed before a subsequent sequence of up to 3 shocks was attempted. This procedure was repeated for a maximum of 3 cycles. ROSC was defined as the return of supraventricular rhythm with a mean aortic pressure above 50 mmHg for a minimum of 5 min. Following ROSC, mechanical ventilation was continued with 100% oxygen for the first hour, 50% for the second hour and 21% thereafter. There was no pharmacology agent used during CPR. After the experiment, all animals were euthanized by an overdose of pentobarbital (150 mg/kg via the femoral artery). Abdominal cavity culture was performed to prove the laparotomy was sterile. A necropsy was performed to document injuries to thoracic, abdominal vessels and viscera caused by surgical intervention. 2.3. Measurements Aortic and right atrial pressures, electrocardiogram and ETCO2 were continuously recorded for 8 h on a personal computer-based data acquisition system (DATAQ Instruments, Akron, OH). Myocardial function, including cardiac output (CO), ejection fraction (EF) and myocardial performance index (MPI), were
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assessed at baseline, 1, 2, 4, 6 and 8 h after ROSC using echocardiography (HD 11 XE, Philips Healthcare, Andover, MA) with a 12.5 MHz transducer.15 Artery blood samples (0.3 ml) were withdrawn at baseline, 2, 4, 6 and 8 h after ROSC for measuring arterial blood gases with the aid of a Stat Profile pHOx analyzer (Nova Biomedical Corporation, Waltham, MA). For the measurement of serum cytokine, blood samples (1.2 ml) were collected at the same time points and immediately centrifuged at 3000 rpm for 10 min. Then, the serum was stored until analysis. The experimental rat received an equivalent volume (1.5 ml) of arterial blood from a donor rat after the blood withdrawal. TNF-␣ and IL-6 concentrations were determined by using ELISA kits (TNF-␣: R&D Systems, Minneapolis, MN; IL-6: Thermo Scientific, Rockford, IL). The experiments were performed following the manufacturer’s instructions. Microcirculations were visualized at baseline, 1, 2, 4, 6 and 8 h after ROSC with the aid of a sidestream dark-field (SDF) imaging device (MicroScan; MicroVision Medical Inc., Amsterdam, Netherlands) with a 5× optical probe. The sublingual region was assessed near the base of the tongue. For the measurements of intestinal microcirculation, a 2–3 cm segment of the jejunum was withdrawn with its neurovascular supply intact and cushioned with warm saline-soaked gauze. The jejunal microcirculation was assessed on the anti-mesenteric aspect of the serosal side. After microcirculatory measurements were taken, the abdominal contents were then returned into the peritoneal cavity and the abdomen was closed in two layers. The muscular layer of the abdominal wall was sutured in a continuous pattern, and the skin incision was closed with wound clips. Three discrete fields were captured with precaution to minimize motion and pressure artifacts. Microvascular images were recorded on a DVD disk using a DVD recorder (Model DMREZ47V; Panasonic AVC Networks, Dalian, China). Individual images were analyzed offline. Microcirculatory flow index (MFI) was quantitated by the method of Boerma et al.16 The image was divided into four quadrants and the predominant type of flow (absent = 0, intermittent = 1, sluggish = 2 and normal = 3) was assessed in the small vessels of each quadrant, which were less than 20 m in diameter. The MFI score represented the average values of four quadrants. Perfused vessel density (PVD) was measured based on the method of De Backer et al.17 Vessel density was calculated as the number of vessels crossing the lines divided by the total length of the lines. Vessel size was measured with a micrometer scale superimposed in the video display. All recordings were analyzed by three independent observers. 2.4. Statistical analyses Measurements were reported as mean ± SD or median (interquartile range) as indicated. Variables were compared with one-way ANOVA or the Kruskal–Wallis test for nonparametric data. Comparisons between time-based measurements within each group were performed with repeated-measurement analysis of variance. If there was a significant difference in the overall comparison of groups, comparisons between any other two groups were made by the Bonferroni test. Linear correlations were calculated using the Pearson correlation coefficient. A value of p < 0.05 was considered significant. 3. Results Baseline hemodynamics, myocardial function and blood analytical measurements did not differ among groups (Table 1). All animals were successfully resuscitated. There was no difference in the amount of the electric current required to induce VF between CPR groups. Body temperature did not differ significantly
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Table 1 Baseline characteristics. Group
Sham control
Body weight, g Heart rate, bpm MAP, mmHg End-tidal CO2 , mmHg Ejection fraction, % Cardiac output, mL/min MPI pH Lactate, mmol/L
510 346 135 41 70.0 107.0 0.71 7.48 0.9
± ± ± ± ± ± ± ± ±
24 21 6 1 1.0 5.2 0.04 0.03 0.3
Short duration of CA 506 356 133 41 69.5 107.3 0.69 7.51 0.8
± ± ± ± ± ± ± ± ±
15 28 10 1 1.5 2.6 0.04 0.03 0.1
Long duration of CA 503 357 136 41 69.5 107.6 0.68 7.50 0.9
± ± ± ± ± ± ± ± ±
14 24 8 1 1.7 4.1 0.03 0.04 0.2
CA, cardiac arrest; MAP, mean aortic pressure; MPI, myocardial performance index. Values are presented as mean ± SD.
among groups during the experiment. There was no significant difference of CPP between the CPR groups during CPR. CPP at the end of CPR was 25.0 ± 3.2 mmHg in the short duration group and was 24.5 ± 2.9 mmHg in the long duration group. The number of defibrillations did not differ significantly (1.4 ± 0.5 vs. 2.1 ± 2.2) between the short and the long duration groups. The post-resuscitation PVD decreased in parallel with the reduced MFI in both intestinal and sublingual sites. In the long duration group, intestinal PVD was significantly reduced from baseline of 19.8 ± 1.7 to 10.1 ± 1.8 n/mm at 8 h post-resuscitation and MFI from 3.0 ± 0.0 to 1.4 ± 0.7 (both p < 0.05 vs. baseline and sham control). Similarly, sublingual PVD was significantly reduced from the baseline of 5.6 ± 0.8 to 2.3 ± 0.6 n/mm at 8 h post-resuscitation and MFI from 3.0 ± 0.0 to 1.3 ± 0.6 (both p < 0.05 vs. baseline and sham control). Contrary to a progressive recovery of both measurements observed in the short duration group, the significant reduction in both PVD and MFI persisted after resuscitation in the long duration group. At 8 h post-resuscitation, PVD and MFI were significantly lower in the long duration group than in the short duration group: intestinal PVD (10.1 ± 1.8 vs. 17.7 ± 2.8 n/mm, p < 0.05); sublingual PVD (2.3 ± 0.6 vs. 4.8 ± 0.9 n/mm, p < 0.05); intestinal MFI (1.4 ± 0.7 vs. 2.4 ± 0.4, p < 0.05); sublingual MFI (1.3 ± 0.6 vs. 2.4 ± 0.5, p < 0.05). The decreases in intestinal microcirculatory blood flow were closely correlated with the reductions of sublingual microcirculatory blood flow (PVD: r = 0.772, p < 0.01; MFI: r = 0.821, p < 0.01) (Fig. 1). Examples of the images of intestinal and sublingual microvasculature, as obtained by the SDF video microscope at 8 h post-resuscitation, are shown in Fig. 2. Myocardial function was significantly impaired in the postresuscitation period in both CPR groups compared with baseline values. Significantly worse post-resuscitation myocardial function was observed after long duration of CA when compared to the short duration of CA group which gradually recovered over time (Fig. 3). Serum cytokine levels in both CPR groups elevated after resuscitation. There was significantly elevated TNF-␣ levels in the long duration group 2 h after resuscitation. In the short duration group, there was a significant elevation only 8 h after resuscitation. However, for IL-6, except in the long duration group at 6 and 8 h post-resuscitation, none were statistically different from the sham control group (Fig. 4). There were significant correlations between MFI, PVD, myocardial function and serum cytokine levels (Table 2). At 8 h post-resuscitation, ETCO2 values in the long duration group were significantly lower than those of the sham control and the short duration group. Plasma lactate levels in the long duration group remained significantly greater when compared with the sham control and the short duration group (p < 0.05). However, there was no significant difference in heart rate or mean aortic pressure (MAP) between the CPR groups (Table 3).
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Fig. 1. Changes of microcirculatory variables and the correlation between sublingual and intestinal microcirculation. BL, baseline; CA, cardiac arrest; MFI, microcirculatory flow index; PVD, perfused vessel density. *p < 0.05, vs. sham control group. # p < 0.05 vs. short duration of CA group.
4. Discussion The present study demonstrated that both intestinal and sublingual microcirculatory blood flow decreased significantly with increasing duration of cardiac arrest and resuscitation. Intestinal microcirculatory dysfunction is closely correlated with the severity of post-resuscitation syndrome as measured by myocardial Table 2 Correlational analysis between myocardial function, microcirculatory parameters and serum cytokine levels. Sublingual
Intestinal
PVD
MFI
PVD
MFI
Cardiac function CO EF MPI
0.827* 0.846* −0.794*
0.846* 0.823* −0.775*
0.821* 0.767* −0.750*
0.839* 0.818* −0.757*
Cytokine levels TNF-␣ IL-6
−0.332* −0.415*
−0.380* −0.394*
−0.420* −0.406*
−0.307* −0.326*
PVD, perfused vessel density; MFI, microcirculatory flow index; CO, cardiac output; EF, ejection fraction; MPI, myocardial performance index; TNF-␣, tumor necrosis factor-alpha; IL-6, interleukin-6. * p < 0.01
function and inflammatory cytokine levels. Sublingual microcirculatory changes were closely correlated with that of intestinal. In humans, the easiest accessible site to detect microcirculation is the sublingual area. Sublingual microcirculation is considered as a surrogate measure for splanchnic blood flow. Under conditions of circulatory shock, noninvasive sublingual capnometry yielded measurements that were interchangeable with those of gastric tonometry.18–20 Using a direct visualization approach, our findings demonstrated the similar behavior of sublingual and intestinal microcirculation at the early stage after ROSC. Our findings are
Table 3 Characteristics at 8 h post-resuscitation. Group
Sham control
Heart rate, bpm MAP, mmHg End-tidal CO2 , mmHg pH Lactate, mmol/L
337 126 40 7.54 1.1
± ± ± ± ±
27 4 1 0.02 0.2
Short duration of CA 319 104 36 7.54 1.3
± ± ± ± ±
51 13* 2* 0.04 0.4
Long duration of CA 342 97 29 7.51 2.4
± ± ± ± ±
28 17* 6* , # 0.09 0.9* , #
CA, cardiac arrest; MAP indicates mean aortic pressure; Values are presented as mean ± SD. * p < 0.05 vs. sham control group. # p < 0.05 vs. short duration of CA group.
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Fig. 2. Images of sublingual and intestinal microcirculation obtained by the sidestream dark-field (SDF) video microscope at 8 h post-resuscitation.
consistent with earlier studies not only of circulatory shock but also of endotoxic and septic shock, which indicates that the severity and the time course of microcirculatory changes were similar in the sublingual and gut region.21–24 Interestingly, two studies on sepsis demonstrated conflicting findings on the relationship between these two sites. Though both revealed time-varying correlations between these two sites, a clinical study claimed that microcirculatory changes were not parallel on day 1 while another animal study demonstrated that the initial strong correlation disappeared over time.25,26 Highly heterogeneous nature of the sepsis victims might explain the conflicting results. Additionally, both studies applied therapeutic interventions that may further influence the intestinal microcirculation.27,28 Although post-resuscitation abnormalities mimic the disorders observed in sepsis, cardiac dysfunction is one of the primary distinguishing characteristics. The impairment of cardiac function during the post-resuscitation period can precipitate a downward splanchnic blood flow. However, microcirculatory changes can be dispatched from changes in regional blood flow and differ among visceral organs especially in low-flow states.29 In the present study, we revealed a close correlation between cardiac function and microcirculatory changes in sublingual and intestinal sites in the early stage following resuscitation. Noticeably, in the long duration of the CA group, cardiac function deteriorated over time, intestinal microcirculatory parameters experienced a slightly upward trend after PR 1 h and then dropped to approximately 50% of baseline value at PR 8 h. The autoregulation may account for the fluctuation of microcirculatory blood flow. In fact, the regulation of microcirculatory blood flow in the intestine wall is extremely complicated. Not only systemic factors but also local factors such as inflammatory cytokines, vasomotor function and metabolic products affect the intestinal microcirculation. Under this experimental condition, we speculate that the intestinal microcirculatory dysfunction caused by systemic insults was beyond the limit of the autoregulatory threshold.
The severity of intestinal microcirculatory dysfunction was magnified when the duration of cardiac arrest and resuscitation was prolonged. In the present study, intestinal microcirculation was impaired in both CPR groups. A partially reversible injury within 8 h was observed in the short duration group. This pattern of response was consistent with previous studies that demonstrated a transition from reversible to irreversible intestinal damage as duration of ischemia prolonged.30 In Korth et al.’s study, 4 min of cardiac arrest caused a resembled reduction of intestinal blood flow during the following 60 min. Though they did not observe blood flow thereafter, this phenomenon that the intestinal metabolic changes were gradually restored over time may indicate a resembled recovery of intestinal microcirculation.4 There may be several factors contributing to the fact that prolonged cardiac arrest leads to more severe intestinal microcirculatory derangement. First, as mentioned above, prolonged CA results in more pronounced myocardial dysfunction, the intestinal microcirculation is therefore more likely to be injured as a result of reduced cardiac output. Second, within 4–6 min of CA, high-energy phosphates become depleted, thus favor disruption of microvascular endothelial cells.31 Third, longer duration of CA may result in local intestinal thrombosis and leukocyte plugging, thus causes the decrease in capillary flow.32 Intestinal barrier integrity plays a pivotal role in the development of systemic inflammatory response syndrome, sepsis and multiple organ failure. The intestine is not only a vulnerable target to ischemia but a possible secondary source of inflammatory cytokines as well. We cannot rule out the possibility that postresuscitation disturbed intestinal microcirculation may trigger an inflammatory cascade. IL-6 as well as TNF-␣ are key mediators of the systemic inflammatory response. Our results confirmed an intense increase of such cytokines in the early hours after CA.7 The decreases of microcirculatory blood flow were correlated with the increases of inflammatory cytokine levels. In our study, a discrepant time course of TNF-␣ and IL-6 levels after different durations of CA
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Fig. 4. Serum TNF-␣ and IL-6 concentrations at baseline and post-resuscitation (mean ± SD). *p < 0.05, vs. sham control group. # p < 0.05 vs. short duration of CA group.
Fig. 3. Myocardial function at baseline and post-resuscitation. EF, ejection fraction; CO, cardiac output; MPI, myocardial performance index; BL, baseline; CA, cardiac arrest; VF, ventricular fibrillation. Values are presented as mean ± SD. *p < 0.05, vs. sham control group. # p < 0.05 vs. short duration of CA group.
were observed. The TNF-␣ level remained at high values 2 h after ROSC in the long duration of the CA group while there was a 6 h lag after short duration. IL-6 expression, which can be induced by TNF␣, elevated at 6 h post-resuscitation after 8 min of CA. Although the present study was not designed to compare survival rates among groups, several studies have confirmed high levels of TNF-␣ and IL-6 could discriminate between survivors and nonsurvivors.7,33 Clinically, intestine injuries after cardiac arrest are often underestimated though it might be a potentially devastating complication even following successful resuscitation.34 The manifestations may be nonspecific and delayed. The direct in vivo observation of the intestinal microcirculation is not clinically feasible and making early detections is therefore difficult. Our study demonstrated that the changes of sublingual microcirculation during post-resuscitation reflected changes of intestinal microcirculation, myocardial function and inflammatory cytokine levels. This suggests that sublingual microcirculation may therefore provide a new option to monitor post-resuscitation patients at the bedside. To interpret the results of our experimental study, it is necessary to take several limitations into consideration. First, therapeutic intervention was not applied in the study, which is different from
clinical situations. Microcirculation can be affected by several factors. The use of epinephrine, hypothermia or fluid therapy may have reduced differences in microcirculation parameters between groups. Second, we investigated the microvasculature of serosa layer rather than the mucosa layer. It is likely that microcirculatory changes in these two sites are different. Third, we didn’t investigate the neurologic function which is one important component of post-resuscitation syndrome. Fourth, using SDF imaging, PVD and MFI were evaluated in a semiquantitative way. A quantitative analysis might generate a more accurate outcome. Fifth, movement and pressure artifacts hinder the analysis of the images though we tried to reduce the interference. Finally, we did not measure the abdominal pressures which might affect the microcirculatory blood flow in the intestinal wall.35 However, a sham control group was enrolled to rule out this factor. 5. Conclusion The severity of post-resuscitation intestinal microcirculatory dysfunction is closely correlated with that of myocardial function and inflammatory cytokine levels. The measurement of sublingual microcirculation reflects changes of intestinal microcirculation and may therefore provide a new option for post-resuscitation monitoring. Conflict of interest None of the authors have any conflict of interest to report. Acknowledgement Lisa Luna contributed to the editing of this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.resuscitation. 2014.02.019.
J. Qian et al. / Resuscitation 85 (2014) 833–839
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