严重失血性休克大鼠液体复苏后的免疫修复

Immune recovery after fluid resuscitation in rats with severe hemorrhagic shock 严重失血性休克大鼠液体复苏后的免疫修复 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:luyuanqiang201@hotmail.comluyuanqiang@zju.edu.cn †E-mail:luyuanqiang201@hotmail.comluyuanqiang@zju.edu.cn luyuanqiang201@hotmail.com 1 2 2 Department of Emergency Medicine, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Emergency Medicine, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Cancer Institute, Zhejiang Tumor Hospital, Hangzhou 310022, China Cancer Institute, Zhejiang Tumor Hospital, Hangzhou 310022, China Objective: 2 To investigate the effects of resuscitation with normal saline (NS), hypertonic saline (HTS), and hydroxyethyl starch (HES) on regulatory T cells (Tregs), helper T 1 (Th1)/Th2 and cytotoxic T 1 (Tc1)/Tc2 profiles in the treatment of hemorrhagic shock. Methods: 2 Rats subjected to severe hemorrhagic shock were resuscitated for 30 min with NS ( n =8), HTS ( n =8), or HES ( n =8); sham ( n =8) and naive control ( n =8) groups were used for comparison. Following fluid resuscitation, the whole shed blood was reinfused for 30 min, and the rats were observed with continuous hemodynamic monitoring for 120 min. CD4 + CD25 + Foxp3 + Treg proportions, Th1/Th2 and Tc1/Tc2 profiles in spleen were analyzed by three-color flow cytometry. Results: 2 The proportion of CD4 + CD25 + Foxp3 + Tregs and ratios of Th1/Th2 and Tc1/Tc2 did not differ among control, sham, and HTS groups, but were significantly lower in NS and HES groups (both P < 0.05 vs. sham); NS and HES levels were similar. The level of Tc1 was significantly increased in HTS ( P < 0.05 vs. sham), and levels of Tc2 were increased in NS, HES, and HTS groups compared to sham (all P < 0.05), but did not differ from each other. Conclusions: 2 HTS resuscitation has a greater impact on immune system recovery than NS or HES by preserving the proportion of Tregs and maintaining the balance between Th1/Th2 and Tc1/Tc2 cells in the spleen. Thus, HTS resuscitation provides potential immunomodulatory activity in the early stage after hemorrhagic shock. 目的 2 严重失血性休克大鼠模型早期阶段使用不同的液体复苏，比较脾脏组织中调节性T细胞（Tregs）、辅助性T细胞1（Th1）/Th2以及细胞毒性T细胞1（Tc1）/Tc2的不同变化，初步探讨其免疫修复机制。 创新点 2 （1）脾脏为机体重要免疫器官，检测其中的免疫细胞变化，比外周血更具敏感性和特异性；（2）将免疫反应中多环节的免疫细胞变化进行协同分析，结果更具创新性和科学性，为临床上形成规范的救治方案提供了科学的实践资料。 方法 2 将SD雄性大鼠随机分成5组，其中对照组和Sham组(假手术)仅作为比较，其余三组在建立严重失血性休克大鼠模型后，采用不同的液体复苏:等渗盐水(NS组)、高渗盐水(HTS组)和羟乙基淀粉(HES组)。然后再灌注30分钟，并持续监测血液动力学120分钟，最后心脏穿刺，取脾脏组织，通过三色荧光标记流式细胞术进一步分析CD4 + CD25 + Foxp3 + Treg细胞含量，以及Th1/Th2和Tc1/Tc2的比值。 结论 2 液体复苏后大鼠脾脏中CD4 + CD25 + Foxp3 + Tregs细胞含量、Th1/Th2和Tc1/Tc2的比值在对照组、Sham组和HTS组中无差异，并都显著高于NS组和HES组。与Sham组比较，HTS组中Tc1水平明显升高，而NS组、HES组和HTS组中Tc2水平均有升高，且三组之间Tc2水平无差别。因此，对于维持脾脏中Treg细胞含量、Th1/Th2和Tc1/Tc2平衡的作用上，HTS液体复苏对免疫系统的影响大于NS和HES。综上所述，在失血性休克后的早期阶段HTS复苏可提供潜在的免疫修复作用。 Regulatory T cells Helper T cells Cytotoxic T cells Hemorrhagic shock 调节性T细胞 辅助性T细胞 细胞毒性T细胞 失血性休克 Introduction 1 Hemorrhagic shock is an emergency event primarily caused by low organ perfusion, which results in marked and widespread oxidative tissue damage. Crucial treatment of this condition involves timely control of hemorrhage and adequate intravascular volume replacement. Even with successful fluid resuscitation, acute respiratory distress syndrome, systemic inflammatory response syndrome, or sepsis and multiple organ dysfunction syndrome can develop. These are the leading causes of death in middle and late phases of hemorrhagic shock. These conditions are thought to result from hyperinflammation or immunodepression (Menger and Vollmar, 2004 2004 ; Moore et al., 2004 2004 ; 2006 2006 ; Brøchner and Toft, 2009 2009 ). Fluids for resuscitation include normal saline (NS), hypertonic saline (HTS), and hydroxyethyl starch (HES), although controversy remains on which strategy is superior (Angele et al., 2008 2008 ). Recently, the immunologic effects of resuscitation with HTS solutions have been examined (Yip et al., 2007 2007 ; Murao et al., 2009 2009 ; Vincenzi et al., 2009 2009 ; Isayama et al., 2012 2012 ). HTS stimulates the rapid release of adenosine triphosphate (ATP) in a time-and concentration-dependent manner, which enhances T-cell activation by increasing Ca 2+ influx (Yip et al., 2007 2007 ). In addition, HTS resuscitation suppresses the release of proinflammatory cytokines (Isayama et al., 2012 2012 ), and thus might improve immunosuppressive reactions after hemorrhagic shock. Indeed, our previous studies indicate that HTS alleviates immune disorders and limits tissue injury after hemorrhagic shock (Lu et al., 2007 2007 ; 2008 2008 ; 2010 2010 ). Although serious immune dysfunction often appears in the middle or late stage, immune and inflammation systems are involved from the onset of hemorrhagic shock. Hence, it is essential to reveal the initial impacts of resuscitation with different fluids on the immune system to provide advice for early recovery and immune intervention strategies. The purpose of this study was to compare the effects of three resuscitation fluids on immune cell regulation in the early stage of hemorrhagic shock. Specifically, we evaluated the proportion of regulatory T cells (Tregs), which help maintain immune balance, and can limit inflammatory tissue damage caused by infection, autoimmunity, allogeneic immune responses, and antitumor immunity (Belkaid and Rouse, 2005 2005 ; Zhao et al., 2013 2013 ); we also evaluated the ratios of helper T (Th) and cytotoxic T (Tc), which can execute many crucial immune functions (Marcu et al., 2007 2007 ; Zhang et al., 2008 2008 ; Chen et al., 2014 2014 ). Materials and methods 1 Ethics statement 2 This study was approved by the Ethics Committee of the School of Medicine, Zhejiang University, Hangzhou, China. Animals were handled according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011 2011 ). Animals 2 Forty male Sprague-Dawley rats, weighing 280–330 g, were purchased from the Laboratory Animal Center of the Medical Institute of Zhejiang Province, China and were maintained on standard rat diet and tap water ad libitum before the experiment. Animals were housed under controlled light/dark conditions for at least 3 d; the light period was from 8:00 a.m. to 5:00 p.m. in specific pathogen-free conditions. Experimental protocol 2 Rats were anesthetized with 40 mg/kg pentobarbital intraperitoneally (IP) and placed in a supine position on a warm pad (25 °C). After sterilization with 10% (0.1 g/ml) povidone-iodine solution, the right carotid artery was isolated and cannulated with a polyethylene catheter, which was used for blood withdrawal and connected to a pressure transducer and computerized physiograph system (model 1290C; Hewlett-Packard Co., Palo Alto, CA, USA) for continuous hemodynamic monitoring. In the same way, the left femoral vein was cannulated for fluid infusion, and rats were heparinized (500 U/kg). Rats were observed for 10 min post-cannulation to ensure stable blood hemodynamics and spontaneous breathing. Blood loss during cannulation was collected by gauze for measurement by weight; only rats with <0.22 g blood loss were used for fluid resuscitation experiments. Severe and controlled hemorrhagic shock was induced according to our previous studies (Lu et al., 2007 2007 ; 2008 2008 ; 2010 2010 ). Briefly, while under the mild anesthesia (40 mg/kg pentobarbital IP), acute hemorrhage was initiated (time=0 min) with four controlled blood withdrawals via the right carotid arterial cannula: 10 ml/kg per 5 min for the first two withdrawals, then 5 ml/kg per 5 min for the second two, to yield a total hemorrhage volume of 30 ml/kg. The shed blood was collected in glass syringes with heparin for later reinfusion. Fluid resuscitation was administered after 30 min. In the third stage, at 60 min, rats were reinfused with the whole shed blood to represent in-hospital emergency treatment. An observation stage then ensued (time=90–210 min) via continuous hemodynamic monitoring. Rats were then sacrificed by heart puncture and the spleens were removed for preparation of cell suspensions used in subsequent analyses. Grouping of animals 2 Forty rats were randomly assigned to one of five groups ( n =8 each group). In the control group, rats received no operative intervention. In the sham group, rats only received anesthesia, cannulation, heparinization, and observation. In the fluid resuscitation phase, rats in the NS resuscitation group received 0.9% (9 g/L) NaCl infusion for a 3:1 ratio against the blood loss volume (Gurfinkel et al., 2003 2003 ; Alam et al., 2004 2004 ; Barros et al., 2011 2011 ; Maier and Alam, 2011 2011 ). The HTS group received 6.0 ml/kg body weight of 7.5% (0.075 g/ml) NaCl solution for a 1:5 ratio against blood loss volume (Murao et al., 2003a 2003a ; 2003b 2003b ). The HES group received 30 ml/kg body weight of 6% HES (200/0.5; Fresenius Kabi, Baden Humboldt, Germany) solution for a 1:1 ratio against blood loss volume (Barros et al., 2011 2011 ). Flow cytometry 2 RBC Lysis Buffer was used to remove the red blood cells from the sample. The monoplast suspension of spleen was prepared according to our previous protocol (Lu et al., 2013 2013 ). Spleen cell suspensions were cultured for 4 h at 37 °C at 5% CO 2 in RPMI-1640 media (Sigma-Aldrich, St. Louis, MO, USA) containing phorbol myristate acetate, ionomycin, monensin (Beyotime, China), and fetal bovine serum. Three-color flow cytometer (FC500, FACSCalibur; Beckman-Coulter, Brea, CA, USA) was then used to detect the various cell types. The software FACSCalibur (Beckman-Coulter, USA) was used for flow cytometric analysis. Anti-rat CD4 FITC (fluorescein isothiocyanate), CD25 PE (phycoerythrin), and Foxp3 PerCP-Cy5 (peridinin chlorophyll protein-cyanine 5) antibodies, and their isotype controls (eBioscience, San Diego, CA, USA) were used for detecting Tregs (CD4 + CD25 + Foxp3 + ). Anti-rat CD3 FITC, CD8a PerCP, interleukin (IL)-4 PE, and interferon (IFN)- γ PE antibodies, and their isotype controls (BD Biosciences, San Jose, CA, USA) were used for detecting Th1 (CD3 + IFN- γ + CD8 – ), Th2 (CD3 + IL-4 + CD8 – ), Tc1 (CD3 + IFN- γ + CD8 + ), and Tc2 (CD3 + IL-4 + CD8 + ) cells. Statistical analysis 2 Data analyses were performed using SPSS Version 13.0 software (SPSS Inc., Chicago, IL, USA). Cell type percentages and ratios were compared by the homogeneity test, and then one-way analysis of variance (ANOVA) and the least significant difference t -test were applied for comparisons among the five groups. A two-way repeated-measures ANOVA was performed to compare arterial pressures among the groups. Data are presented as mean±standard deviation (SD) with a P < 0.05 considered statistically significant. Results 1 Mean arterial pressure 2 All rats undergoing acute hemorrhagic shock survived the experiments, with about 50% loss of blood volume based on weight. Mean arterial pressures (MAPs) measured throughout the experiment are presented in Fig. 1 1 . Blood pressure levels for rats in the control group were not obtained, as they did not receive polyethylene catheters for monitoring. Acute hemorrhage initially induced a dramatic decrease in MAP, to about 20 mmHg at 20 min, which increased to about 30 mmHg at 30 min by self-compensation. Fluid resuscitation restored pressures within 15 min, with MAPs approaching 90 mmHg at the 45 min time-point. The MAPs then remained stable throughout the emergency treatment (60–90 min) and observation (90–210 min) stages. MAP levels remained unchanged throughout the entire experiment for rats in the sham group. Sham: sham group ( n =8); NS: normal saline group ( n =8); HTS: hypertonic saline group ( n =8); HES: hydroxyethyl starch group ( n =8). Data are represented as mean±SD A two-way repeated-measures ANOVA was performed with fluids as the between-subjects factors and MAPs obtained at different time-points as within-subject variables. As a Mauchly’s test of sphericity indicated that variances in differences among the groups were not equal ( df =54, P < 0.05), degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity ( ε =0.34). As a result, no significant difference in MAP was found among NS, HTS, and HES groups during the experiment ( F [6.78, 50.82]=1.943, P > 0.05). Changes of CD4 + CD25 + Foxp3 + Tregs in spleen 2 The representative illustration of flow cytometry for Tregs is shown in Fig. 2 2 . The percentage of CD4 + CD25 + Foxp3 + Tregs in rats receiving HTS fluid resuscitation did not differ from those in sham and control rats (Table 1 1 ). However, the percentages of Tregs in NS and HES groups were similar and significantly lower than those in the control and sham groups ( P < 0.05). (a) Upper right quadrant represents CD4 + CD25 + cells. (b) Upper right quadrant represents CD4 + CD25 + Foxp3 + Tregs HES: hydroxyethyl starch; HTS: hypertonic saline; NS: normal saline. Data are represented as mean±SD. P< 0.05, vs. control; P < 0.05, vs. sham; P < 0.05, vs. NS; P < 0.05, vs. HES Group n Tregs (%)  Control 8 2.21±0.35  Sham 8 1.96±0.30  NS 8 1.36±0.19 * * ∆ ∆  HTS 8 2.10±0.31 # # § §  HES 8 1.24±0.34 * * ∆ ∆ 3 F 17.072 P 0.000 Changes of Th1/Th2 and Tc1/Tc2 ratios in spleen 2 The representative illustration of flow cytometry for Th and Tc is shown in Fig. 3 3 . Ratios of Th1/Th2 and Tc1/Tc2 in rats receiving HTS resuscitation did not differ from those in the control and sham rats (Table 2 2 ). However, both ratios were significantly lower in rats receiving NS and HES than in the control and sham groups ( P < 0.05). Interestingly, the percentage of Tc1 cells was significantly higher only in the HTS group, whereas the levels of Tc2 in all three groups receiving fluid resuscitation were equally and significantly improved ( P < 0.05 vs. control and sham; Fig. 4 4 ). (a) Upper right quadrants represent CD3 + interferon (IFN)-γ + (top) and CD3 + interleukin (IL)-4 + (bottom) cells, respectively. (b) Upper right quadrant represents CD3 + IFN-γ + CD8 + Tc1 cells and lower right quadrant represents CD3 + IFN-γ + CD8 − Th1 cells (top); upper right quadrant represents CD3 + IL-4 + CD8 + Tc2 cells and lower right quadrant represents CD3 + IL-4 + CD8 − Th2 cells (bottom) Control: control group ( n =8); Sham: sham group ( n =8); NS: normal saline group ( n =8); HTS: hypertonic saline group ( n =8); HES: hydroxyethyl starch group ( n =8). Data are represented as mean±SD. * P < 0.05, vs. control; ∆ P < 0.05, vs. sham by one-way ANOVA and the least significant difference t -test HES: hydroxyethyl starch; HTS: hypertonic saline; NS: normal saline. Data are represented as mean±SD. P < 0.05, vs. control; P < 0.05, vs. sham; P < 0.05, vs. NS; P < 0.05, vs. HES Group n Th1/Th2 Tc1/Tc2  Control 8 0.37±0.08 0.46±0.10  Sham 8 0.35±0.08 0.47±0.11  NS 8 0.22±0.06 * * ∆ ∆ 0.33±0.04 * * ∆ ∆  HTS 8 0.32±0.07 # # § § 0.42±0.08 # # § §  HES 8 0.23±0.07 * * ∆ ∆ 0.32±0.08 * * ∆ ∆ 4 F 7.051 5.395 P 0.000 0.002 Discussion 1 Tregs mediate immune and inflammatory responses in many pathophysiologic processes. A sufficient quantity of Tregs is critical in keeping inflammation balance and reducing tissue injury (Li et al., 2009 2009 ; Fogle et al., 2010b 2010b ; Tang et al., 2014 2014 ; Zhao et al., 2015 2015 ; Zhang et al., 2016 2016 ). Excessive activity or quantity of Tregs can promote infectious deterioration, sepsis, or tumor immune escape, whereas an insufficient amount can result in overactive inflammatory responses (Burgents et al., 2010 2010 ; Fogle et al., 2010a 2010a ; Carambia et al., 2014 2014 ). Tregs have been shown to be differentially influenced by various resuscitation fluids after hemorrhagic shock (Murao et al., 2009 2009 ; Isayama et al., 2012 2012 ). In the present study, resuscitation with HTS did not alter the percentage of Tregs in the spleen, whereas NS and HES significantly lowered it. Thus, resuscitation with a small volume (1:5 ratio against blood loss volume) of HTS can stabilize spleen Tregs in the early phase, and may play a beneficial role in reducing immunologic stress after hemorrhagic shock. Indeed previous studies have shown that HTS resuscitation can reduce the injury to lungs and intestinal mucosa after hemorrhagic shock (Fernandes et al., 2009 2009 ; Gao et al., 2009 2009 ; Lu et al., 2010 2010 ). The proposed mechanism by which HTS can mediate these effects is by shrinking cells and mechanically altering membranes, resulting in the release of ATP and promotion of T-cell function (Loomis et al., 2003 2003 ; Woehrle et al., 2010 2010 ; Ledderose et al., 2016 2016 ). Whether a similar mechanism is involved in the effect on Tregs in our rat hemorrhagic shock model requires further research. The ratios of Th1/Th2 and Tc1/Tc2 can reflect the balance of immune stability to some degree. Several studies have shown that hemorrhagic shock disturbs this balance by enhancing the differentiations of Th2 and Tc2 cells and suppressing the differentiations of Th1 and Tc1 cells, which increases the risks of sepsis and multiple organ failure (Marcu et al., 2007 2007 ; Miller et al., 2007 2007 ; Chen et al., 2014 2014 ). In the present study, resuscitation with HES and NS significantly lowered the ratios of Th1/Th2 and Tc1/Tc2. However, the quantities of Tc1 and Tc2 cells were increased with HTS, whereas only Tc2 cells increased with NS and HES. It is possible that HTS resuscitation preserves Th1 and Tc1 cell differentiation, and may have more effective immunomodulation activity than NS and HES in the early stage after hemorrhagic shock. Consistent with this, Zhang et al. ( 2008 2008 ) showed that the differentiation of Th1 cells was enhanced after resuscitation with HES. Peripheral blood was not analyzed in the present study in consideration of the effect of blood reinfusion on immune function of peripheral blood mononuclear cells. However, the results of the present study indicated that resuscitation with all three fluids equally restored MAP levels in the early stage of hemorrhagic shock. Although the MAP in rats receiving HTS was slightly lower than those in the other groups at the end of the fluid resuscitation stage, it was still high enough ((97.50±6.83) mmHg) to maintain blood supply to vital organs, based on previous reports (Lu et al., 2010 2010 ; Barros et al., 2011 2011 ; Vallet, 2011 2011 ; Cui et al., 2014 2014 ). Fluid resuscitation with NS or HES may result in hypoosmolar dehydration, thus stimulating an immune system response and inflammatory injury, whereas HTS may promote a more beneficial state of immunologic stress. 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DOI: 10.1631/jzus.B1200293 http://doi.org/10.1631/jzus.B1200293 . β-receptor blocker influences return of spontaneous circulation and chemical examination in rats during cardiopulmonary resuscitation Compliance with ethics guidelines Feng YAO, Yuan-qiang LU, Jiu-kun JIANG, Lin-hui GU, and Han-zhou MOU declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1600370 Project supported by the National Natural Science Foundation of China (No. 81272075) 2016-08-23 2017-04-10 2016-10-17 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 18