American Journal of Emergency Medicine xxx (2015) xxx–xxx
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American Journal of Emergency Medicine journal homepage: www.elsevier.com/locate/ajem
Original Contribution
A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock☆,☆☆ Jingxiang Zhao, PhD a,1, Guocheng Pan, BS b,1, Bo Wang, PhD a, Yuhua Zhang, PhD a, Guoxing You, MD a, Ying Wang, MD a, Dawei Gao, PhD b,⁎, Hong Zhou, PhD a,⁎⁎, Lian Zhao a,⁎⁎ a b
Institute of Transfusion Medicine, Academy of Military Medical Sciences, No. 27th Taiping Road, HaiDian, Beijing, China Department of Biological Engineering, College of Environment and Chemical Engineering, Yanshan University, Qinhuangdao, China
a r t i c l e
i n f o
Article history: Received 22 October 2014 Received in revised form 2 February 2015 Accepted 21 February 2015 Available online xxxx
a b s t r a c t Background: We aimed to evaluate the effects of resuscitation with different ratios of fresh frozen plasma (FFP) to red blood cells (RBCs) on pulmonary inflammatory injury and to illuminate the beneficial effects of FFP on lung protection compared with lactated ringers (LR) using a rat model of hemorrhagic shock. Methods: Rats underwent pressure-controlled hemorrhage for 60 minutes and were then transfused with LR for initial resuscitation. Thereafter, the rats were transfused with varying ratios of FFP:RBC (1:4, 1:2, 1:1, and 2:1) or LR:RBC (1:1) to hold their mean arterial pressure (MAP) at 100 ± 3 mm Hg for 30 minutes. After 4 hours of observation, lung tissue was harvested to determine the wet/dry weight, myeloperoxidase levels, tumor necrosis factor α levels, macrophage inflammatory protein 2 (MIP-2) levels, inducible nitric oxide synthase activity, and the nuclear factor κB p65 DNA-binding activity. Results: With an increase in the FFP:RBC ratio, the volume of required RBC to maintain the target MAP decreased. The MAP value in each group was not significantly different during the whole experiment period. The values of the wet/dry weights and MIP-2 were significantly lower in the FFP:RBC = 1:1 group than the other groups (P b .05). All parameters detected above were predominantly lower in the FFP:RBC = 1:1 group than the FFP:RBC = 1:2 group and the LR:RBC = 1:1 group (P b .05). In addition, all parameter values were lower in the FFP:RBC = 1:1 group than in the FFP:RBC = 2:1 group, but only the wet/dry weight, myeloperoxidase, and MIP-2 values were significantly different (P b .05). Conclusions: Resuscitation with a 1:1 ratio of FFP to RBC results in decreased lung inflammation. Compared with LR, FFP could further mitigate lung inflammatory injury. © 2015 Published by Elsevier Inc.
1. Introduction According to data obtained from both the military and the clinic, the damage control resuscitation (DCR) strategy as a structured intervention begins immediately after rapid initial assessment in the emergency department (ED). Thus, DCR represents a potential opportunity to
☆ Conflicts of interests: On behalf of all authors, the corresponding author states that there is no conflict of interest. ☆☆ Funding: This work was supported by a research grant from the Capital Health Project of Beijing Science and Technology Commission (Z121100005312014) and the National Natural Science Foundation of China (31271001). ⁎ Correspondence to: Dawei Gao, PhD, Department of Biological Engineering, College of Environment and Chemical Engineering, Yanshan University, Qinhuangdao, China. ⁎⁎ Corresponding authors at: Institute of Transfusion Medicine, Academy of Military Medical Sciences, No. 27th Taiping Road, HaiDian, Beijing, China. E-mail addresses: [email protected] (D. Gao), [email protected] (H. Zhou), [email protected] (L. Zhao). 1 The first 2 authors contributed equally to this work.
reduce mortality further. For a severely injured casualty, DCR consists of 2 parts and is initiated within minutes of arrival in the ED. First, resuscitation is limited to keep systolic blood pressure at approximately 80 to 90 mm Hg, which prevents renewed bleeding from recently clotted vessels. Second, intravascular volume restoration is accomplished using thawed plasma as a primary resuscitation fluid in a high ratio with red blood cells (RBCs) [1,2]. Based on previous civilian clinical and military studies, Borgman et al [3,4] reviewed massively transfused (≥ 10 U RBC/24 hours) combatants and demonstrated that the mortality reduction was impressive in patients with a higher FFP:RBC ratio (1:1.4) and that they had a longer median time to death than those with a lower ratio. The study by Holcomb et al [5] examined trauma patients at 16 trauma centers who required massive transfusion, and the study determined that an FFP:packed red blood cell ratio of 1:2 or higher compared with lower ratios was associated with improved 30-day survival. Sperry et al [6] found mortality differences in those patients who received ratios of plasma to packed red blood cell of approximately 1:1.5. Therefore, increasing the ratio of fresh frozen plasma (FFP) to RBCs is beneficial in clinical situations associated with traumatic hemorrhage.
http://dx.doi.org/10.1016/j.ajem.2015.02.041 0735-6757/© 2015 Published by Elsevier Inc.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
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J. Zhao et al. / American Journal of Emergency Medicine xxx (2015) xxx–xxx
However, the currently available evidence supporting higher FFP:RBC ratios for resuscitation is inconclusive. The present civilian clinical and military studies are not randomized controlled trials that account for survivorship bias [7,8]. The survivorship bias arises because the patients who were not expected to live long enough to receive FFP were categorized (without randomization) to the low FFP:RBC cohorts in observational studies. However, patients surviving long enough to receive sufficient FFP were categorized to the high FFP:RBC cohorts [7]. Fresh frozen plasma is usually used as a volume expander and is currently indicated for the management and prevention of bleeding in coagulopathic patients [1,8]. Recently, Letourneau et al [9,10] demonstrated that treatment with FFP stabilized the endothelium. The overall beneficial effect of FFP may be decreased endothelium permeability, which could contribute to vascular instability, edema, and other detrimental effects of injury and resuscitation, despite increased leukocyteendothelial binding. Therefore, the beneficial effects of FFP on lung tissue protection besides coagulopathy should be further investigated. In the present study, we examined the effect of transfusing various ratios of FFP:RBC on lung injury with the goal of identifying the appropriate FFP:RBC ratio for DCR. In addition, we aimed to find the association between FFP with transfusion-related lung injury in a rat model of hemorrhagic shock. 2. Materials and methods All animal studies were approved by the Ethics Committee of the Institute of Transfusion Medicine, Academy of Military Medical Sciences. The care and handling of the animals were in accordance with the National Institutes of Health guidelines. All efforts were made to minimize the number of animals used and animal suffering. 2.1. Animal preparation Fifty-six male Wistar rats (280-300 g; Vital River Laboratories, Beijing, China) were anesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital (Peking Chemical Agent Co, Peking, China) and placed on a warming pad (TMS-202; Softron, Beijing, China) in a supine position that was maintained at 37 ± 0.1°C throughout the experiment [11]. Supplemental doses of 10 mg/kg sodium pentobarbital were given per hour after the start of resuscitation. The left carotid artery, right femoral artery, and vein were exposed, isolated, and cannulated with polyethylene catheters (PE-50). Four hundred units per kilogram heparin (Chinese Medicine Group Chemical Agent Co, Peking, China) was administered intravenously to inhibit the coagulation of blood in the experimental equipment. The left carotid artery was used for continuous monitoring of blood pressure. The right femoral artery was used to achieve bleeding using a withdrawal pump (LZS-AJ10; Softron) and to sample the arterial blood for blood gas analysis. The right femoral vein was used for the administration of fluids.
2.2. Collection of blood components Donor male rats were anesthetized and heparinized. The femoral artery was isolated and cannulated to obtain the whole blood. Anticoagulated whole blood was centrifuged at 1000g for 10 minutes immediately after collection to obtain plasma and fresh RBC. Plasma was stored at −80°C to prepare the FFP for future use. Fresh RBC was gently mixed with thawed FFP in the ratio specified for blood transfusion. 2.3. Experimental protocols Fig. 1 depicts the experimental protocol. After the operation and instrumentation, the rat hemorrhagic shock model was prepared as described previously [12] with several modifications. A volumecontrolled hemorrhage of 21 mL/kg (~35% of total blood volume) was performed for 20 minutes through the right femoral arterial catheter, and the animals were stabilized for 10 minutes. Subsequently, the animals were subjected to a slower hemorrhage of 10 mL/kg (~10% of total blood volume) for 15 minutes, and hemorrhaging was continued as needed to maintain a MAP at 50 ± 5 mm Hg for 15 minutes. The total amount of blood withdrawn was recorded. Resuscitation with lactated ringers (LR) (equal to the maximum blood volume withdrawn) was performed, and the animals were stabilized for 30 minutes (R”). Subsequently, all of the rats were divided into 5 groups (n = 8/group) and were resuscitated with the varying ratios of FFP (LR) and RBC to hold their MAP at 100 ± 3 mm Hg for 30 minutes (R0): (1) an FFP to RBC ratio of 1:4 (1:4), (2) an FFP to RBC ratio of 1:2 (1:2), (3) an FFP to RBC ratio of 1:1 (1:1), (4) an FFP to RBC ratio of 2:1 (2:1), and (5) an LR to RBC ratio of 1:1 (LR1:1). All infusions were performed using a pump driven at a constant rate of 0.33 mL/min in all groups. Thereafter, all rats were monitored for 240 minutes, and the MAP was recorded at intervals of 30 minutes (R30, R60, R90, R120, R150, R180, and R240). Sham animals underwent cannulation and anesthesia for an identical period as the resuscitation animals but were not bled (sham) (n = 8). Shock animals underwent hemorrhage but not resuscitation (shock) (n = 8). At the end of the observation period, the animals were sacrificed by exsanguination, and the lungs were quickly removed. The right lung was excised for the wet to dry weight ratio. The left lung was washed with cold saline, snap frozen in liquid nitrogen, and stored in liquid nitrogen for further processing. 2.4. Hemodynamic and blood gas analysis Hemodynamic parameters, such as MAP, heart rate, and rectal temperature, were recorded using a multiple-channel recorder (MP150 Research System; Biopac System Inc, Montreal, Canada). Blood gas analysis was performed at baseline (base), after blood withdrawal (shock) and 4 hours after resuscitation using 0.15 mL of arterial blood with a blood gas analyzer (ABL90 FLEX; Radiometer, Copenhagen, Denmark).
Fig. 1. Study design.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
J. Zhao et al. / American Journal of Emergency Medicine xxx (2015) xxx–xxx
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2.5. Wet/dry weight ratio
2.8. Nuclear factor κB activity
The freshly harvested right lungs from all rats were weighed and placed in the vacuum drying oven for 5 days at 60°C. The lungs were weighed again when they were dry. The values are expressed as the ratio of wet-to-dry weight [13].
The pulmonary nuclear factor κB (NF-κB) activity was measured as the nuclear translocation and DNA binding of the p65 subunit. We used 30 μg of whole-cell extract from tissues to detect protein with a commercially available enzyme-linked immunosorbent assay (TransAM NF-κB p65; Active Motif, Carlsbad, CA). The whole tissue extract (30 μg) was added to the plate and incubated for 1 hour at room temperature. After washing the plate, a primary antibody identifying activated p65 was added and incubated for another 1 hour. An anti– immunoglobulin G horseradish peroxidase conjugate was later added to the plate. The color was developed according to the manufacturer's instructions. The optical density value at 450 nm was measured on a plate reader [14].
2.6. Myeloperoxidase and inducible nitric oxide synthase activities Snap-frozen lung tissues were homogenized and sonicated on ice in 0.9% saline. The homogenates were centrifuged at 1500g for 15 minutes at 4°C. The supernatants were used for the measurement of MPO and inducible nitric oxide synthase (iNOS) activities (Jiancheng Biological Institute, Nanjing, China) using colorimetric determination according to the manufacturer's recommendations as we previously reported [12].
2.9. Statistical analysis 2.7. Inflammatory cytokines The pulmonary levels of tumor necrosis factor α (TNF-α) and macrophage inflammatory protein 2 (MIP-2) were determined using enzymelinked immunosorbent assay kits (Fangcheng Biotechnology, Beijing, China) according to the manufacturer's instructions. Briefly, the upper lobe of the left lung was homogenized and sonicated on ice in 0.9% saline. The homogenates were centrifuged at 1500g for 15 minutes at 4°C, and the supernatants were assayed for TNF-α and MIP-2. The cytokine values are expressed as nanogram per liter protein.
The data are presented as the means ± SDs. The data were analyzed using a one-way analysis of variance followed by a Student-Newman-Keuls test where appropriate. When normality and homogeneity of variance assumptions were not satisfied, a nonparametric Kruskal-Wallis test was applied. The Student t test was used to compare the differences between the FFP:RBC = 1:1 group and the LR:RBC = 1:1. The significance level was P b .05. A commercial software package (SAS Institute Inc, Cary, NC) was used for data analysis.
Fig. 2. Shed blood volume and the required blood volume for maintaining the target MAP (100 ± 3 mmHg). (A) Shed blood percentage of total blood volume (%), (B) The amount of blood transfusion of blood loss percentage (%), (C) Red blood cell transfusion volume accounts for the percentage of the amount of blood transfusion (%), (D) The hemoglobin levels at the end of blood transfusion (R0). The data are presented as the means ± standard deviation. *Pb0.05 versus the FFP:RBC=1:4 group; **Pb0.05 versus the FFP:RBC=1:2 group; #Pb0.05 versus the FFP:RBC=1:1 group.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
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J. Zhao et al. / American Journal of Emergency Medicine xxx (2015) xxx–xxx
Fig. 3. Mean arterial pressures (MAP) during experiments. The data are presented as the means ± standard deviation. The MAP of rats undergoing hemorrhage and resuscitation with ratios of FFP (LR) to RBC including 2:1, 1:1, 1:2, 1:4 and LR1:1 compared with sham rats, *Pb0.05 versus other groups..
3.2. Pulmonary wet/dry weight ratio, MPO activity, and MIP-2 levels
3. Results
3.1. Basic characteristics All rats were alive during the experiment. The average shed blood volume was 54.03% ± 2.01% of the estimated circulating blood (60 mL/kg) volume, and there were no significant differences among the resuscitation groups (P N .05) (Fig. 2A). However, with the increase in the FFP:RBC ratio from the 2:1 ratio of FFP:RBC to the 1:2 ratio of FFP:RBC, the total volume of FFP and RBC required to maintain the target MAP (100 ± 3 mm Hg) gradually increased. The 1:4 ratio of FFP:RBC required a greater transfusion volume to hold the target MAP than the 1:2 ratio of FFP:RBC (Fig. 2B). With the increase in the FFP:RBC ratio, the volume of required RBC and the hemoglobin levels at the end of the blood transfusion (R0) had a tendency to decrease (Fig. 2C and D). Compared with the FFP:RBC = 1:1 group, the LR:RBC = 1:1 group exhibited a similar volume of total FFP/RBC transfusion and RBC transfusion (Fig. 2). All resuscitation groups were equivalent at baseline and had a similar MAP value at the end of hemorrhage and LR resuscitation. After resuscitation with varying ratios of FFP:RBC or LR:RBC to hold the same target MAP, the MAP was maintained, and there were no significant differences among groups throughout the observation period (P N .05). Still, the MAP in all hemorrhaged groups was significantly lower than in the sham group throughout the experiments (Fig. 3). All resuscitation groups exhibited no significant difference with regard to arterial PO2, PCO2, pH, bicarbonate, base excess, and lactate at the end of observation (Table).
Rats resuscitated with a 1:1 ratio of FFP to RBC exhibited significantly decreased wet/dry weight ratios compared with all other resuscitation groups (P b .05, Fig. 4A). Lung tissue MPO activity was significantly increased in the shock group compared with the sham group and was significantly attenuated after resuscitation. The MPO activity in the FFP:RBC = 1:1 group was significantly lower than in the other resuscitation groups, except for the FFP:RBC = 1:4 group (P b .05, Fig. 4B). Lung MIP-2 levels were significantly elevated in the shock group compared with the sham group and were significantly attenuated after resuscitation. The MIP-2 levels were prominently lower in the FFP:RBC = 1:1 group compared with the other resuscitation groups (P b .05, Fig. 4C). 3.3. Pulmonary inflammatory cytokines and NF-κB activity Lung TNF-α levels were elevated in the shock group compared with the sham group. Lung TNF-α levels in the FFP:RBC = 1:1 group were lower than in the other resuscitation groups but were only significantly different compared with the FFP:RBC = 1:2 group and the LR:RBC = 1:1 group (P b .05, Fig. 5A). The lung iNOS and NF-κB activities were significantly different between the hemorrhage and sham groups. The lung iNOS and NF-κB activities in the FFP:RBC = 1:1 group were lower than in the other resuscitation groups but were only statistically significantly different compared with the FFP:RBC = 1:2 group and the LR:RBC = 1:1 group (P b .05, Fig. 5B and C).
Table Arterial blood gas analysis of resuscitation groups in the end of observation
PO2 (mm Hg) PCO2 (mm Hg) pH Bicarbonate (mmol/L) Base excess Lactate (mmol/L)
FFP:RBC = 1:4
FFP:RBC = 1:2
FFP:RBC = 1:1
FFP:RBC = 2:1
LR:RBC = 1:1
P
88.77 ± 6.81 31.56 ± 3.46 7.472 ± 0.027 23.46 ± 4.55 −1.48 ± 4.39 1.82 ± 0.71
88.53 ± 7.33 28.45 ± 5.32 7.481 ± 0.022 23.60 ± 2.85 −0.83 ± 1.72 1.92 ± 0.38
92.77 ± 4.82 29.26 ± 4.08 7.484 ± 0.021 23.82 ± 2.30 −0.83 ± 2.10 1.85 ± 0.31
89.12 ± 3.82 31.99 ± 1.78 7.497 ± 0.013 24.54 ± 0.93 0.75 ± 0.40 1.90 ± 0.69
86.31 ± 6.37 33.98 ± 3.51 7.463 ± 0.021 24.72 ± 2.68 −1.10 ± 3.47 1.90 ± 0.53
.4228 .1632 .0749 .9031 .8492 .8728
Data are presented as means ± SD. P values were obtained by comparing between groups.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
J. Zhao et al. / American Journal of Emergency Medicine xxx (2015) xxx–xxx
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Fig. 4. Lung wet/dry ratio, MPO and MIP-2 activity. Lung wet/dry ratio after hemorrhagic shock and resuscitation (A), lung MPO activity (B), and MIP-2 levels in tissue homogenates at the end of the experiment (C). The data are presented as the means ± SD. εP b .05 vs the sham group; δP b .05 vs the shock group; ⁎P b .05 vs the FFP:RBC = 1:4 group; ⁎⁎P b .05 vs the FFP:RBC = 1:2 group; #P b .05 vs the FFP:RBC = 1:1 group.
4. Discussion According to the DCR strategies using a high ratio of FFP to RBC [1], we adapted a rodent model of controlled hemorrhage to evaluate the effects of transfusing various ratios of FFP to RBC on the early inflammatory response, and the beneficial effect of FFP on lung protection compared with LRs was also investigated. The present study showed that resuscitation with a 1:1 ratio of FFP to RBC suppressed pulmonary edema and pulmonary inflammatory responses compared with other ratios of FFP to RBC. Resuscitation with a high ratio of FFP to RBC would require a greater volume of RBC transfusion to maintain MAP. Furthermore, the use of FFP in DCR strategies could significantly decrease pulmonary inflammation and pulmonary edema compared with LR. With the development of transfusion strategies, the DCR was considered to be effective resuscitation methods for the treatment of severe hemorrhage in civilian and military scenarios. In our study, the experimental protocol was designed according to the DCR method. Lactated ringers were transfused during initial resuscitation for treating hemorrhagic shock, and then blood components were transfused to hold the target MAP. As a result, although the volumes of the total blood components required to maintain the target MAP were significantly different among the resuscitation groups, the MAP levels were similar throughout the observation period, and arterial blood gas parameters including PO2, PCO2, pH, HCO3−, base excess, and lactate were not different at the end of the observation period. These data indicate that the different resuscitation strategies used here did not significantly differ in their hemodynamic effects and acid-base equilibrium. Hemorrhagic shock and resuscitation frequently induce systemic inflammatory responses, and the lung is the first targeted organ. Lung injury after hemorrhagic shock is associated with localized inflammation due to the accumulation of activated neutrophils and increased edema resulting from the increase in microvascular permeability. In
this article, the value of the pulmonary wet/dry weight ratios in the FFP:RBC = 1:1 group was significantly lower than other resuscitation groups, and coincidently, the arterial PO2 value, which was affected by lung edema in the FFP:RBC = 1:1 group, was also higher than the other groups even though the difference was not statistically significant (Table). Macrophage inflammatory protein 2 is a murine chemokine and has been shown to play crucial roles in regulating neutrophil migration in animal injury models. Myeloperoxidase (MPO) activity correlates with the sequestration and activation of neutrophils in lung tissue, and it is elevated in murine acute lung injury [15]. Lomas et al [16] showed that antibody neutralization of MIP-2 resulted in decreased MPO activity and less histologic lung injury. In our study, resuscitation with a 1:1 ratio of FFP to RBC demonstrated a marked reduction in MIP-2 levels, which correlated with reduced MPO activity and pulmonary edema. Thus, the above results showed that resuscitation with a 1:1 ratio of FFP to RBC produced minor lung injury. Nuclear factor κB is a major proinflammatory transcription factor that is activated early after hemorrhage and resuscitation and is involved in regulating the expression of genes including inflammatory cytokines, chemokines, and adhesion molecules that participate in inflammation [17]. Tumor necrosis factor α is an essential cytokine in the cascade that causes lung endothelial injury. Robert Shenkar and Edward Abraham [18] showed that the enhanced expression of TNF-α and MIP-2 in lung neutrophils is dependent on xanthine oxidase, which is involved in the activation of NF-κB in the lungs after hemorrhage. In the present study, we observed that the trend of NF-κB expression in the various FFP to RBC groups was similar to our results for the expression of MIP-2 and TNF-α. In addition, iNOS can be expressed in many different cell types, including stromal or parenchymal cells and inflammatory cells, during the hemorrhagic shock. Furthermore, iNOS activity is essential for the up-regulation of the inflammatory response and participates in end-organ damage in the lungs during hemorrhagic shock [19-21]. Harkin et al [22] indicated that TNF-α levels
Fig. 5. Levels of inflammatory parameters in the lungs at the end of the experiments. Tumor necrosis factor α (A), iNOS (B), and NF-κB (C). The data are presented as the means ± SD. εP b .05 vs the sham group; δP b .05 vs the shock group; ⁎P b .05 vs the FFP:RBC = 1:4 group; ⁎⁎P b .05 vs the FFP:RBC = 1:2 group; #P b .05 vs the FFP:RBC = 1:1 group.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
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J. Zhao et al. / American Journal of Emergency Medicine xxx (2015) xxx–xxx
are significantly attenuated by treatment with the iNOS inhibitor N(6)(iminoethyl)-lysine. Similarly, resuscitation with a 1:1 ratio of FFP to RBC in our study demonstrated a reduction in TNF-α levels and was correlated with reduced iNOS levels. On the whole, although not all of the inflammatory parameters detected above had significant differences between the 1:1 ratio of FFP to RBC group and the other resuscitation groups, there was a coincidental tendency of lower values in the above parameters for the 1:1 ratio of FFP to RBC group, which suggested resuscitation with a 1:1 ratio of FFP to RBC would mitigate pulmonary inflammatory injury. Implementation of DCR for hemorrhagic shock treatment in civilian trauma centers has been controversial due to the effects associated with plasma transfusion [23,24]. Based on the results of various ratio groups, we used LR instead of FFP in the 1:1 ratio of FFP to RBC group to investigate whether the use of FFP in DCR might have beneficial effects on lung protection besides coagulopathy. In this study, compared with LR, resuscitation with FFP cannot significantly improve the MAP and decrease the total and RBC volumes during resuscitation, but resuscitation with FFP significantly mitigated lung edema, inflammatory responses, and neutrophil activation.
5. Conclusions The current experimental data indicate that resuscitation with a 1:1 ratio of FFP to RBC after hemorrhagic shock attenuated the inflammatory response and edema in the lung compared with other ratios. In addition, the use of FFP in DCR strategies could mitigate pulmonary inflammatory injury compared with LR.
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Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
Contents lists available at ScienceDirect
American Journal of Emergency Medicine journal homepage: www.elsevier.com/locate/ajem
Original Contribution
A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock☆,☆☆ Jingxiang Zhao, PhD a,1, Guocheng Pan, BS b,1, Bo Wang, PhD a, Yuhua Zhang, PhD a, Guoxing You, MD a, Ying Wang, MD a, Dawei Gao, PhD b,⁎, Hong Zhou, PhD a,⁎⁎, Lian Zhao a,⁎⁎ a b
Institute of Transfusion Medicine, Academy of Military Medical Sciences, No. 27th Taiping Road, HaiDian, Beijing, China Department of Biological Engineering, College of Environment and Chemical Engineering, Yanshan University, Qinhuangdao, China
a r t i c l e
i n f o
Article history: Received 22 October 2014 Received in revised form 2 February 2015 Accepted 21 February 2015 Available online xxxx
a b s t r a c t Background: We aimed to evaluate the effects of resuscitation with different ratios of fresh frozen plasma (FFP) to red blood cells (RBCs) on pulmonary inflammatory injury and to illuminate the beneficial effects of FFP on lung protection compared with lactated ringers (LR) using a rat model of hemorrhagic shock. Methods: Rats underwent pressure-controlled hemorrhage for 60 minutes and were then transfused with LR for initial resuscitation. Thereafter, the rats were transfused with varying ratios of FFP:RBC (1:4, 1:2, 1:1, and 2:1) or LR:RBC (1:1) to hold their mean arterial pressure (MAP) at 100 ± 3 mm Hg for 30 minutes. After 4 hours of observation, lung tissue was harvested to determine the wet/dry weight, myeloperoxidase levels, tumor necrosis factor α levels, macrophage inflammatory protein 2 (MIP-2) levels, inducible nitric oxide synthase activity, and the nuclear factor κB p65 DNA-binding activity. Results: With an increase in the FFP:RBC ratio, the volume of required RBC to maintain the target MAP decreased. The MAP value in each group was not significantly different during the whole experiment period. The values of the wet/dry weights and MIP-2 were significantly lower in the FFP:RBC = 1:1 group than the other groups (P b .05). All parameters detected above were predominantly lower in the FFP:RBC = 1:1 group than the FFP:RBC = 1:2 group and the LR:RBC = 1:1 group (P b .05). In addition, all parameter values were lower in the FFP:RBC = 1:1 group than in the FFP:RBC = 2:1 group, but only the wet/dry weight, myeloperoxidase, and MIP-2 values were significantly different (P b .05). Conclusions: Resuscitation with a 1:1 ratio of FFP to RBC results in decreased lung inflammation. Compared with LR, FFP could further mitigate lung inflammatory injury. © 2015 Published by Elsevier Inc.
1. Introduction According to data obtained from both the military and the clinic, the damage control resuscitation (DCR) strategy as a structured intervention begins immediately after rapid initial assessment in the emergency department (ED). Thus, DCR represents a potential opportunity to
☆ Conflicts of interests: On behalf of all authors, the corresponding author states that there is no conflict of interest. ☆☆ Funding: This work was supported by a research grant from the Capital Health Project of Beijing Science and Technology Commission (Z121100005312014) and the National Natural Science Foundation of China (31271001). ⁎ Correspondence to: Dawei Gao, PhD, Department of Biological Engineering, College of Environment and Chemical Engineering, Yanshan University, Qinhuangdao, China. ⁎⁎ Corresponding authors at: Institute of Transfusion Medicine, Academy of Military Medical Sciences, No. 27th Taiping Road, HaiDian, Beijing, China. E-mail addresses: [email protected] (D. Gao), [email protected] (H. Zhou), [email protected] (L. Zhao). 1 The first 2 authors contributed equally to this work.
reduce mortality further. For a severely injured casualty, DCR consists of 2 parts and is initiated within minutes of arrival in the ED. First, resuscitation is limited to keep systolic blood pressure at approximately 80 to 90 mm Hg, which prevents renewed bleeding from recently clotted vessels. Second, intravascular volume restoration is accomplished using thawed plasma as a primary resuscitation fluid in a high ratio with red blood cells (RBCs) [1,2]. Based on previous civilian clinical and military studies, Borgman et al [3,4] reviewed massively transfused (≥ 10 U RBC/24 hours) combatants and demonstrated that the mortality reduction was impressive in patients with a higher FFP:RBC ratio (1:1.4) and that they had a longer median time to death than those with a lower ratio. The study by Holcomb et al [5] examined trauma patients at 16 trauma centers who required massive transfusion, and the study determined that an FFP:packed red blood cell ratio of 1:2 or higher compared with lower ratios was associated with improved 30-day survival. Sperry et al [6] found mortality differences in those patients who received ratios of plasma to packed red blood cell of approximately 1:1.5. Therefore, increasing the ratio of fresh frozen plasma (FFP) to RBCs is beneficial in clinical situations associated with traumatic hemorrhage.
http://dx.doi.org/10.1016/j.ajem.2015.02.041 0735-6757/© 2015 Published by Elsevier Inc.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
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However, the currently available evidence supporting higher FFP:RBC ratios for resuscitation is inconclusive. The present civilian clinical and military studies are not randomized controlled trials that account for survivorship bias [7,8]. The survivorship bias arises because the patients who were not expected to live long enough to receive FFP were categorized (without randomization) to the low FFP:RBC cohorts in observational studies. However, patients surviving long enough to receive sufficient FFP were categorized to the high FFP:RBC cohorts [7]. Fresh frozen plasma is usually used as a volume expander and is currently indicated for the management and prevention of bleeding in coagulopathic patients [1,8]. Recently, Letourneau et al [9,10] demonstrated that treatment with FFP stabilized the endothelium. The overall beneficial effect of FFP may be decreased endothelium permeability, which could contribute to vascular instability, edema, and other detrimental effects of injury and resuscitation, despite increased leukocyteendothelial binding. Therefore, the beneficial effects of FFP on lung tissue protection besides coagulopathy should be further investigated. In the present study, we examined the effect of transfusing various ratios of FFP:RBC on lung injury with the goal of identifying the appropriate FFP:RBC ratio for DCR. In addition, we aimed to find the association between FFP with transfusion-related lung injury in a rat model of hemorrhagic shock. 2. Materials and methods All animal studies were approved by the Ethics Committee of the Institute of Transfusion Medicine, Academy of Military Medical Sciences. The care and handling of the animals were in accordance with the National Institutes of Health guidelines. All efforts were made to minimize the number of animals used and animal suffering. 2.1. Animal preparation Fifty-six male Wistar rats (280-300 g; Vital River Laboratories, Beijing, China) were anesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital (Peking Chemical Agent Co, Peking, China) and placed on a warming pad (TMS-202; Softron, Beijing, China) in a supine position that was maintained at 37 ± 0.1°C throughout the experiment [11]. Supplemental doses of 10 mg/kg sodium pentobarbital were given per hour after the start of resuscitation. The left carotid artery, right femoral artery, and vein were exposed, isolated, and cannulated with polyethylene catheters (PE-50). Four hundred units per kilogram heparin (Chinese Medicine Group Chemical Agent Co, Peking, China) was administered intravenously to inhibit the coagulation of blood in the experimental equipment. The left carotid artery was used for continuous monitoring of blood pressure. The right femoral artery was used to achieve bleeding using a withdrawal pump (LZS-AJ10; Softron) and to sample the arterial blood for blood gas analysis. The right femoral vein was used for the administration of fluids.
2.2. Collection of blood components Donor male rats were anesthetized and heparinized. The femoral artery was isolated and cannulated to obtain the whole blood. Anticoagulated whole blood was centrifuged at 1000g for 10 minutes immediately after collection to obtain plasma and fresh RBC. Plasma was stored at −80°C to prepare the FFP for future use. Fresh RBC was gently mixed with thawed FFP in the ratio specified for blood transfusion. 2.3. Experimental protocols Fig. 1 depicts the experimental protocol. After the operation and instrumentation, the rat hemorrhagic shock model was prepared as described previously [12] with several modifications. A volumecontrolled hemorrhage of 21 mL/kg (~35% of total blood volume) was performed for 20 minutes through the right femoral arterial catheter, and the animals were stabilized for 10 minutes. Subsequently, the animals were subjected to a slower hemorrhage of 10 mL/kg (~10% of total blood volume) for 15 minutes, and hemorrhaging was continued as needed to maintain a MAP at 50 ± 5 mm Hg for 15 minutes. The total amount of blood withdrawn was recorded. Resuscitation with lactated ringers (LR) (equal to the maximum blood volume withdrawn) was performed, and the animals were stabilized for 30 minutes (R”). Subsequently, all of the rats were divided into 5 groups (n = 8/group) and were resuscitated with the varying ratios of FFP (LR) and RBC to hold their MAP at 100 ± 3 mm Hg for 30 minutes (R0): (1) an FFP to RBC ratio of 1:4 (1:4), (2) an FFP to RBC ratio of 1:2 (1:2), (3) an FFP to RBC ratio of 1:1 (1:1), (4) an FFP to RBC ratio of 2:1 (2:1), and (5) an LR to RBC ratio of 1:1 (LR1:1). All infusions were performed using a pump driven at a constant rate of 0.33 mL/min in all groups. Thereafter, all rats were monitored for 240 minutes, and the MAP was recorded at intervals of 30 minutes (R30, R60, R90, R120, R150, R180, and R240). Sham animals underwent cannulation and anesthesia for an identical period as the resuscitation animals but were not bled (sham) (n = 8). Shock animals underwent hemorrhage but not resuscitation (shock) (n = 8). At the end of the observation period, the animals were sacrificed by exsanguination, and the lungs were quickly removed. The right lung was excised for the wet to dry weight ratio. The left lung was washed with cold saline, snap frozen in liquid nitrogen, and stored in liquid nitrogen for further processing. 2.4. Hemodynamic and blood gas analysis Hemodynamic parameters, such as MAP, heart rate, and rectal temperature, were recorded using a multiple-channel recorder (MP150 Research System; Biopac System Inc, Montreal, Canada). Blood gas analysis was performed at baseline (base), after blood withdrawal (shock) and 4 hours after resuscitation using 0.15 mL of arterial blood with a blood gas analyzer (ABL90 FLEX; Radiometer, Copenhagen, Denmark).
Fig. 1. Study design.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
J. Zhao et al. / American Journal of Emergency Medicine xxx (2015) xxx–xxx
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2.5. Wet/dry weight ratio
2.8. Nuclear factor κB activity
The freshly harvested right lungs from all rats were weighed and placed in the vacuum drying oven for 5 days at 60°C. The lungs were weighed again when they were dry. The values are expressed as the ratio of wet-to-dry weight [13].
The pulmonary nuclear factor κB (NF-κB) activity was measured as the nuclear translocation and DNA binding of the p65 subunit. We used 30 μg of whole-cell extract from tissues to detect protein with a commercially available enzyme-linked immunosorbent assay (TransAM NF-κB p65; Active Motif, Carlsbad, CA). The whole tissue extract (30 μg) was added to the plate and incubated for 1 hour at room temperature. After washing the plate, a primary antibody identifying activated p65 was added and incubated for another 1 hour. An anti– immunoglobulin G horseradish peroxidase conjugate was later added to the plate. The color was developed according to the manufacturer's instructions. The optical density value at 450 nm was measured on a plate reader [14].
2.6. Myeloperoxidase and inducible nitric oxide synthase activities Snap-frozen lung tissues were homogenized and sonicated on ice in 0.9% saline. The homogenates were centrifuged at 1500g for 15 minutes at 4°C. The supernatants were used for the measurement of MPO and inducible nitric oxide synthase (iNOS) activities (Jiancheng Biological Institute, Nanjing, China) using colorimetric determination according to the manufacturer's recommendations as we previously reported [12].
2.9. Statistical analysis 2.7. Inflammatory cytokines The pulmonary levels of tumor necrosis factor α (TNF-α) and macrophage inflammatory protein 2 (MIP-2) were determined using enzymelinked immunosorbent assay kits (Fangcheng Biotechnology, Beijing, China) according to the manufacturer's instructions. Briefly, the upper lobe of the left lung was homogenized and sonicated on ice in 0.9% saline. The homogenates were centrifuged at 1500g for 15 minutes at 4°C, and the supernatants were assayed for TNF-α and MIP-2. The cytokine values are expressed as nanogram per liter protein.
The data are presented as the means ± SDs. The data were analyzed using a one-way analysis of variance followed by a Student-Newman-Keuls test where appropriate. When normality and homogeneity of variance assumptions were not satisfied, a nonparametric Kruskal-Wallis test was applied. The Student t test was used to compare the differences between the FFP:RBC = 1:1 group and the LR:RBC = 1:1. The significance level was P b .05. A commercial software package (SAS Institute Inc, Cary, NC) was used for data analysis.
Fig. 2. Shed blood volume and the required blood volume for maintaining the target MAP (100 ± 3 mmHg). (A) Shed blood percentage of total blood volume (%), (B) The amount of blood transfusion of blood loss percentage (%), (C) Red blood cell transfusion volume accounts for the percentage of the amount of blood transfusion (%), (D) The hemoglobin levels at the end of blood transfusion (R0). The data are presented as the means ± standard deviation. *Pb0.05 versus the FFP:RBC=1:4 group; **Pb0.05 versus the FFP:RBC=1:2 group; #Pb0.05 versus the FFP:RBC=1:1 group.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
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Fig. 3. Mean arterial pressures (MAP) during experiments. The data are presented as the means ± standard deviation. The MAP of rats undergoing hemorrhage and resuscitation with ratios of FFP (LR) to RBC including 2:1, 1:1, 1:2, 1:4 and LR1:1 compared with sham rats, *Pb0.05 versus other groups..
3.2. Pulmonary wet/dry weight ratio, MPO activity, and MIP-2 levels
3. Results
3.1. Basic characteristics All rats were alive during the experiment. The average shed blood volume was 54.03% ± 2.01% of the estimated circulating blood (60 mL/kg) volume, and there were no significant differences among the resuscitation groups (P N .05) (Fig. 2A). However, with the increase in the FFP:RBC ratio from the 2:1 ratio of FFP:RBC to the 1:2 ratio of FFP:RBC, the total volume of FFP and RBC required to maintain the target MAP (100 ± 3 mm Hg) gradually increased. The 1:4 ratio of FFP:RBC required a greater transfusion volume to hold the target MAP than the 1:2 ratio of FFP:RBC (Fig. 2B). With the increase in the FFP:RBC ratio, the volume of required RBC and the hemoglobin levels at the end of the blood transfusion (R0) had a tendency to decrease (Fig. 2C and D). Compared with the FFP:RBC = 1:1 group, the LR:RBC = 1:1 group exhibited a similar volume of total FFP/RBC transfusion and RBC transfusion (Fig. 2). All resuscitation groups were equivalent at baseline and had a similar MAP value at the end of hemorrhage and LR resuscitation. After resuscitation with varying ratios of FFP:RBC or LR:RBC to hold the same target MAP, the MAP was maintained, and there were no significant differences among groups throughout the observation period (P N .05). Still, the MAP in all hemorrhaged groups was significantly lower than in the sham group throughout the experiments (Fig. 3). All resuscitation groups exhibited no significant difference with regard to arterial PO2, PCO2, pH, bicarbonate, base excess, and lactate at the end of observation (Table).
Rats resuscitated with a 1:1 ratio of FFP to RBC exhibited significantly decreased wet/dry weight ratios compared with all other resuscitation groups (P b .05, Fig. 4A). Lung tissue MPO activity was significantly increased in the shock group compared with the sham group and was significantly attenuated after resuscitation. The MPO activity in the FFP:RBC = 1:1 group was significantly lower than in the other resuscitation groups, except for the FFP:RBC = 1:4 group (P b .05, Fig. 4B). Lung MIP-2 levels were significantly elevated in the shock group compared with the sham group and were significantly attenuated after resuscitation. The MIP-2 levels were prominently lower in the FFP:RBC = 1:1 group compared with the other resuscitation groups (P b .05, Fig. 4C). 3.3. Pulmonary inflammatory cytokines and NF-κB activity Lung TNF-α levels were elevated in the shock group compared with the sham group. Lung TNF-α levels in the FFP:RBC = 1:1 group were lower than in the other resuscitation groups but were only significantly different compared with the FFP:RBC = 1:2 group and the LR:RBC = 1:1 group (P b .05, Fig. 5A). The lung iNOS and NF-κB activities were significantly different between the hemorrhage and sham groups. The lung iNOS and NF-κB activities in the FFP:RBC = 1:1 group were lower than in the other resuscitation groups but were only statistically significantly different compared with the FFP:RBC = 1:2 group and the LR:RBC = 1:1 group (P b .05, Fig. 5B and C).
Table Arterial blood gas analysis of resuscitation groups in the end of observation
PO2 (mm Hg) PCO2 (mm Hg) pH Bicarbonate (mmol/L) Base excess Lactate (mmol/L)
FFP:RBC = 1:4
FFP:RBC = 1:2
FFP:RBC = 1:1
FFP:RBC = 2:1
LR:RBC = 1:1
P
88.77 ± 6.81 31.56 ± 3.46 7.472 ± 0.027 23.46 ± 4.55 −1.48 ± 4.39 1.82 ± 0.71
88.53 ± 7.33 28.45 ± 5.32 7.481 ± 0.022 23.60 ± 2.85 −0.83 ± 1.72 1.92 ± 0.38
92.77 ± 4.82 29.26 ± 4.08 7.484 ± 0.021 23.82 ± 2.30 −0.83 ± 2.10 1.85 ± 0.31
89.12 ± 3.82 31.99 ± 1.78 7.497 ± 0.013 24.54 ± 0.93 0.75 ± 0.40 1.90 ± 0.69
86.31 ± 6.37 33.98 ± 3.51 7.463 ± 0.021 24.72 ± 2.68 −1.10 ± 3.47 1.90 ± 0.53
.4228 .1632 .0749 .9031 .8492 .8728
Data are presented as means ± SD. P values were obtained by comparing between groups.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
J. Zhao et al. / American Journal of Emergency Medicine xxx (2015) xxx–xxx
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Fig. 4. Lung wet/dry ratio, MPO and MIP-2 activity. Lung wet/dry ratio after hemorrhagic shock and resuscitation (A), lung MPO activity (B), and MIP-2 levels in tissue homogenates at the end of the experiment (C). The data are presented as the means ± SD. εP b .05 vs the sham group; δP b .05 vs the shock group; ⁎P b .05 vs the FFP:RBC = 1:4 group; ⁎⁎P b .05 vs the FFP:RBC = 1:2 group; #P b .05 vs the FFP:RBC = 1:1 group.
4. Discussion According to the DCR strategies using a high ratio of FFP to RBC [1], we adapted a rodent model of controlled hemorrhage to evaluate the effects of transfusing various ratios of FFP to RBC on the early inflammatory response, and the beneficial effect of FFP on lung protection compared with LRs was also investigated. The present study showed that resuscitation with a 1:1 ratio of FFP to RBC suppressed pulmonary edema and pulmonary inflammatory responses compared with other ratios of FFP to RBC. Resuscitation with a high ratio of FFP to RBC would require a greater volume of RBC transfusion to maintain MAP. Furthermore, the use of FFP in DCR strategies could significantly decrease pulmonary inflammation and pulmonary edema compared with LR. With the development of transfusion strategies, the DCR was considered to be effective resuscitation methods for the treatment of severe hemorrhage in civilian and military scenarios. In our study, the experimental protocol was designed according to the DCR method. Lactated ringers were transfused during initial resuscitation for treating hemorrhagic shock, and then blood components were transfused to hold the target MAP. As a result, although the volumes of the total blood components required to maintain the target MAP were significantly different among the resuscitation groups, the MAP levels were similar throughout the observation period, and arterial blood gas parameters including PO2, PCO2, pH, HCO3−, base excess, and lactate were not different at the end of the observation period. These data indicate that the different resuscitation strategies used here did not significantly differ in their hemodynamic effects and acid-base equilibrium. Hemorrhagic shock and resuscitation frequently induce systemic inflammatory responses, and the lung is the first targeted organ. Lung injury after hemorrhagic shock is associated with localized inflammation due to the accumulation of activated neutrophils and increased edema resulting from the increase in microvascular permeability. In
this article, the value of the pulmonary wet/dry weight ratios in the FFP:RBC = 1:1 group was significantly lower than other resuscitation groups, and coincidently, the arterial PO2 value, which was affected by lung edema in the FFP:RBC = 1:1 group, was also higher than the other groups even though the difference was not statistically significant (Table). Macrophage inflammatory protein 2 is a murine chemokine and has been shown to play crucial roles in regulating neutrophil migration in animal injury models. Myeloperoxidase (MPO) activity correlates with the sequestration and activation of neutrophils in lung tissue, and it is elevated in murine acute lung injury [15]. Lomas et al [16] showed that antibody neutralization of MIP-2 resulted in decreased MPO activity and less histologic lung injury. In our study, resuscitation with a 1:1 ratio of FFP to RBC demonstrated a marked reduction in MIP-2 levels, which correlated with reduced MPO activity and pulmonary edema. Thus, the above results showed that resuscitation with a 1:1 ratio of FFP to RBC produced minor lung injury. Nuclear factor κB is a major proinflammatory transcription factor that is activated early after hemorrhage and resuscitation and is involved in regulating the expression of genes including inflammatory cytokines, chemokines, and adhesion molecules that participate in inflammation [17]. Tumor necrosis factor α is an essential cytokine in the cascade that causes lung endothelial injury. Robert Shenkar and Edward Abraham [18] showed that the enhanced expression of TNF-α and MIP-2 in lung neutrophils is dependent on xanthine oxidase, which is involved in the activation of NF-κB in the lungs after hemorrhage. In the present study, we observed that the trend of NF-κB expression in the various FFP to RBC groups was similar to our results for the expression of MIP-2 and TNF-α. In addition, iNOS can be expressed in many different cell types, including stromal or parenchymal cells and inflammatory cells, during the hemorrhagic shock. Furthermore, iNOS activity is essential for the up-regulation of the inflammatory response and participates in end-organ damage in the lungs during hemorrhagic shock [19-21]. Harkin et al [22] indicated that TNF-α levels
Fig. 5. Levels of inflammatory parameters in the lungs at the end of the experiments. Tumor necrosis factor α (A), iNOS (B), and NF-κB (C). The data are presented as the means ± SD. εP b .05 vs the sham group; δP b .05 vs the shock group; ⁎P b .05 vs the FFP:RBC = 1:4 group; ⁎⁎P b .05 vs the FFP:RBC = 1:2 group; #P b .05 vs the FFP:RBC = 1:1 group.
Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
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J. Zhao et al. / American Journal of Emergency Medicine xxx (2015) xxx–xxx
are significantly attenuated by treatment with the iNOS inhibitor N(6)(iminoethyl)-lysine. Similarly, resuscitation with a 1:1 ratio of FFP to RBC in our study demonstrated a reduction in TNF-α levels and was correlated with reduced iNOS levels. On the whole, although not all of the inflammatory parameters detected above had significant differences between the 1:1 ratio of FFP to RBC group and the other resuscitation groups, there was a coincidental tendency of lower values in the above parameters for the 1:1 ratio of FFP to RBC group, which suggested resuscitation with a 1:1 ratio of FFP to RBC would mitigate pulmonary inflammatory injury. Implementation of DCR for hemorrhagic shock treatment in civilian trauma centers has been controversial due to the effects associated with plasma transfusion [23,24]. Based on the results of various ratio groups, we used LR instead of FFP in the 1:1 ratio of FFP to RBC group to investigate whether the use of FFP in DCR might have beneficial effects on lung protection besides coagulopathy. In this study, compared with LR, resuscitation with FFP cannot significantly improve the MAP and decrease the total and RBC volumes during resuscitation, but resuscitation with FFP significantly mitigated lung edema, inflammatory responses, and neutrophil activation.
5. Conclusions The current experimental data indicate that resuscitation with a 1:1 ratio of FFP to RBC after hemorrhagic shock attenuated the inflammatory response and edema in the lung compared with other ratios. In addition, the use of FFP in DCR strategies could mitigate pulmonary inflammatory injury compared with LR.
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Please cite this article as: Zhao J, et al, A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock, Am J Emerg Med (2015), http://dx.doi.org/10.1016/j.ajem.2015.02.041
