Emergency (****); * (*): *-* This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Copyright © 2015 Shahid Beheshti University of Medical Sciences. All rights reserved. Downloaded from: www.jemerg.com 150 Emergency (2015); 3 (4): 150-154 ORIGINAL RESEARCH The Effect of Blood Loss in the Presence and Absence of Severe Soft Tissue Injury on Hemodynamic and Metabolic Parameters; an Experimental study Ali Mohammad Moradi1, Omid Aj1, Shahram Paydar1*, Farzaneh Ketabchi2, Seyed Mostafa Sheid Moosavi2, Shahram Bolandparvaz1, Hamid Reza Abassi1, Aryan Dokht Tamadon3, Davood Mehrabani3 1- Department of Surgery, Trauma Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. 2- Department of Physiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. 3- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. *Corresponding Author: Shahram Paydar, MD. Trauma Research Center, Shahid Rajaei Trauma Hospital, Shahid Chamran blvd, Shiraz, Iran. Tel/Fax: +987116254206. Email: paydarsh@sums.ac.ir Received: November 2014; Accepted: January 2015 Abstract Introduction: The effect of severe soft tissue injury on the severity of hemorrhagic shock is still unknown. There- fore, the present study was aimed to determine hemodynamic and metabolic changes in traumatic/hemorrhagic shock in an animal model. Methods: Forty male rats were randomly divided into 4 equal groups including sham, hemorrhagic shock, soft tissue injury, and hemorrhagic shock + soft tissue injury groups. The changes in blood pressure, central venous pressure (CVP) level, acidity (pH), and base excess were dynamically monitored and com- paredsented. Results: Mean arterial blood pressure decreased significantly in hemorrhagic shock (df: 12; F=10.9; p<0.001) and severe soft tissue injury + hemorrhagic shock (df: 12; F=11.7; p<0.001) groups 15 minutes and 5 minutes after injury, respectively. A similar trend was observed in CVP in severe soft tissue injury + hemorrhagic shock group (df: 12; F=8.9; p<0.001). After 40 minutes, pH was significantly lower in hemorrhagic shock (df: 12; F=6.8; p=0.009) and severe soft tissue injury + hemorrhagic shock (df: 12; F=7.9; p=0.003) groups. Base excess changes during follow ups have a similar trend. (df: 12; F=11.3; p<0.001). Conclusion: The results of this study have shown that the effect of hemorrhage on the decrease of mean arterial blood pressure, CVP, pH, and base excess is the same in the presence or absence of soft tissue injury. Key words: Shock, hemorrhagic; soft tissue injuries; hemodynamics Cite This Article as: Moradi AM, Aj O, Paydar Sh, et al. The effect of blood loss in the presence and absence of severe soft tissue injury on hemodynamic and metabolic parameters in an experimental rat model. Emergency. 2015;3(4):150-4. Introduction: emorrhage is responsible for about 40% of trauma deaths, 33% to 56% of which happen during the pre-hospital period (1). On average, 1 in every 4 patients affected by severe trauma, suffers from coagulopathy. Coagulopathy and severe hemor- rhage plays an important role in determining the final outcome of the patient, their mortality and disability (2- 4). Soft tissue injury is one of the most important injuries that can lead to coagulopathy (5-9) as it can activate both the coagulation pathway and the fibrinolytic system (10). But the effect of severe tissue injury on the severity of hemorrhagic shock is still not known. Only in one study it has been demonstrated that the existence of se- vere tissue injury can decrease the cardiovascular re- sponses in shock and correct bradycardia and hypoten- sion to some extent (11). But other studies have ex- pressed that the physiologic responses observed in trau- matic hemorrhagic shock are no different from other kinds of hemorrhagic shock (12-14). To realize the pathophysiology of traumatic/hemorrhagic shock, reductionistic approaches such as animal models were used to study the physiology of “normal” states, to compare with pathologic conditions to follow the altera- tions after modulation of pathologic states (15-17). A traumatic/hemorrhagic shock animal model was used to simulate the pathophysiological changes characterizing the disease entity in trauma patients with hypovolemic shock (18-22). Therefore, this study was carried out to de- termine hemodynamic and metabolic changes in trau- matic/hemorrhagic shock rat model which can be a clini- cal testing to clarify the process in human patients. Methods: Study design and setting H This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Copyright © 2015 Shahid Beheshti University of Medical Sciences. All rights reserved. Downloaded from: www.jemerg.com 151 Moradi et al The present experimental study was conducted in ac- cordance with the guidelines of ethics for the evaluation of pain in conscious animals. Animal selection, experi- ments, subsequent care and the sacrifice method were all in line with the guidelines of Animal Care Committee of Iran Veterinary Organization. All experiments were performed under aseptic conditions in Laboratory Ani- mal Center of Shiraz University of Medical Sciences. The study was approved by the ethics committee of the insti- tution. The duration of trials and the number of animals used were reduced to a least possible to minimize animal pain and suffering. During the experiments, the animals were kept one per cage, maintained under controlled en- vironmental conditions (21±2ºC, 65–70% relative hu- midity; 12 hour light/dark cycle started at 7:00 Am) and had free access to water and food. Before the experi- ments, all the animals were deprived of food for 4 hours, but had free access to water to prevent excessive dehy- dration during starvation. At the end of the procedure, the rats were euthanized under anesthesia or carbon di- oxide. Animals 40 male Sprague-Dawley experimental rat models (weight, 200-250 gram) were randomly divided into 4 equal groups: sham (catheter insertions were done, without shock induction), hemorrhagic shock, severe soft tissue injury, and severe soft tissue injury + hemor- rhagic shock. Animals that died during surgical proce- dures and rats that lost more than 0.2 mL blood during catheterization or did not have spontaneous breathing 10 minutes after the procedure were excluded. Interventions In all procedures, rats were anesthetized with 60 mg/kg intra-peritoneal pentobarbital (Sigma chemical com- pany, St, Louis, USA) with a 10 mg/kg as maintenance dose (23). Body temperature during anesthesia, cathe- terization, hemorrhage shock, and soft tissue injury was controlled by a heat pad and preserved at 37.0°C. A) Catheterization After the confirmation of deep sedation (no response to noxious stimulus) and stability of respiratory rate, three catheters were inserted into femoral artery, carotid ar- tery, and jugular vein (tip of this catheter were placed in the right atrium) for assessing the arterial blood gas sample, blood pressure, and central venous pressure, re- spectively. In addition, the hemorrhagic shock was in- duced from the femoral catheter. Lidocaine (5mg/kg subcutaneously) was locally used be- fore placing the catheters. Catheterization was done with polyethylene tubes number 50 (PE50) cannula (Becton Dickinson, Sparks, MD; United States). Carotid artery, and jugular vein catheters were connected to pressure transducers and blood pressure analyzer (Micro-Med, Louisville, KY; United States) for continuous monitoring of animals’ hemodynamic state (24). All the catheters were fixed in place using surgical silk sutures. After cath- eterization, all animals were allowed to stabilize (30 minutes) and baseline hemodynamic parameters were assessed and blood samples were taken (0.2 mL, by in- sulin syringes). B) Inducing the hemorrhagic shock After the stabilization of animals and baseline assess- ment, hemorrhagic shock was induced using a fixed- pressure model. For this purpose, the researcher re- moved 1 mL blood every minute through the femoral ar- tery catheter, using a 2-mL syringe (Pars syringe, Teh- ran, Iran). The blood removing was done five times with 10 minute intervals for tuning blood pressure. Bleeding was continued until the mean arterial blood pressure reached 25-30 mmHg. Heparin (500 U/kg) was intrave- nously injected through tail vein to prevent blood clot- ting (24). C) Soft tissue injury A close femoral bone fracture and soft tissue injury with no hemorrhage (contusion model) were induced using a blunt guillotine ramming system with a dropped steel weight (0.5kilogram; from a drop height of 15cm). To en- sure postoperative stabilization, intramedullary stabili- zation was performed using a steel pin (24). Outcome During the experiment, the changes in blood pressure (BP), central vain pressure (CVP), blood acidity (pH), and base excess (BE) were dynamically monitored during 60 minutes. Statistical analysis Data were analyzed using SPSS software (Version 13, Chicago, IL, USA) and were expressed as mean ± SEM. Within-group comparisons were performed by re- peated-measures analysis of variance (ANOVA) and in- ter-group comparisons were done using two-way ANOVA and Tukey post hoc test. P value of less than 0.05 was considered statistically significant. Results: Mean arterial blood pressure significantly dropped in the hemorrhagic shock (df: 12; F=10.9; p<0.001) and se- vere soft tissue injury + hemorrhagic shock (df: 12; F=11.7; p<0.001) groups 15 minutes and 5 minutes after injury, respectively, and it continued until 60 minutes. This decrease was significant compared to the sham (p<0.001) and soft tissue injury (p<0.001) groups (Fig- ure 1). A similar trend was observed in CVP in the severe soft tissue injury + hemorrhagic shock group (df: 12; F=8.9; p<0.001) and in the hemorrhagic shock (df: 12; F=6.4; p<0.001). CVP was significantly reduced from 35 minutes after injury (df: 12; F=7.6; p=0.005). This de- crease was significant compared to the sham (p<0.001) and soft tissue injury (p<0.001) groups (Figure 2). After 40 minutes, pH was significantly lower in the hem- orrhagic shock (df: 12; F=6.8; p=0.009) and severe soft This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Copyright © 2015 Shahid Beheshti University of Medical Sciences. All rights reserved. Downloaded from: www.jemerg.com Emergency (2015); 3 (4): 150-154 152 tissue injury + hemorrhagic shock (df: 12; F=7.9; p=0.003) groups compared to the sham group (Figure 3). Base excess changes during follow ups show a similar trend. Compared to the sham animals, hemorrhagic shock (df: 12; F=11.3; p < 0.001) and severe soft tissue injury + hemorrhagic shock (df: 12; F=10.1; p < 0.001) induction lead to significant decrease in base excess in 40 minutes and 60 minutes after injury (Figure 4). Discussion: The results of this study have shown that the effect of hemorrhage on the decrease of mean arterial blood pres- sure, central vain pressure, blood acidity, and base ex- cess is the same in the presence or absence of soft tissue injury. In this regard, we can say that soft tissue injury in the absence of hemorrhagic shock has no effect on hemo- dynamic and metabolic parameters of the blood. One of the major causes of mortality in traumatic pa- tients, is hemorrhagic shock. Therefore, evaluating the factors affecting the severity of the shock is of great im- portance. In the current study, the presence of severe soft tissue injury was studied as a factor that potentially affects hemorrhagic shock. The results from this study showed that severe tissue injury has no effect on blood pressure, central vain pressure, blood acidity, and base excess. While Little et al. express that the presence of se- vere soft tissue injury can influence the cardiovascular * * * * * * * * * * # # # # # # # # # # # # 0 20 40 60 80 100 120 140 160 0 5 10 15 20 25 30 35 40 45 50 55 60 M e a n r te r ia l b lo o d p r e s s u r e ( m m H g ) Time (minute) Sham sever soft tissue injury Hemorrhagic shock Sever soft tissue injury+ Hemorrhagic shock Figure1: Comparison of mean arterial blood pressure between the experimented groups. *, shows significant difference of hem- orrhagic shock group with the sham group at the same time. #, shows significant difference of severe soft tissue injury + hemor- rhagic shock group with the sham group at the same time. # * * * * * # # # # * # # # # # -10 -8 -6 -4 -2 0 2 4 0 5 10 15 20 25 30 35 40 45 50 55 60 M e a n b lo o d p r e s s u r e ( m m H g ) Time (minute) Sham sever soft tissue injury Hemorrhagic shock Sever soft tissue injury+ Hemorrhagic shock Figure 2: Comparison of central venous pressure between the experimented groups. *, shows significant difference of hemor- rhagic shock group with the sham group at the same time. #, shows significant difference of severe soft tissue injury + hemor- rhagic shock group with the sham group at the same time. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Copyright © 2015 Shahid Beheshti University of Medical Sciences. All rights reserved. Downloaded from: www.jemerg.com 153 Moradi et al Figure3: Comparison of blood pH between the experimented groups. *, shows significant difference of hemorrhagic shock group with the sham group at the same time. #, shows signifi- cant difference of severe soft tissue injury + hemorrhagic shock group with the sham group at the same time. response to hemorrhagic shock (11). The reason for this difference might be that Little et al. had used ischemia to induce the injuries while most soft tissue injuries in- duced by crush injury. The mechanisms involved in these models are different and probably that is the reason for the difference seen in the cardiovascular response be- tween the present study and the Little et al. study (24). Our model of blood withdrawal in the current study combined with active bleeding could produce a severe and reproducible hemorrhagic shock. To date, many hemorrhagic shock–resuscitation models have been es- tablished, but none of them covered the important as- pects regarding severity, clinical reality, and reproduci- bility (25-27). Our method described here and our ex- perimental findings have led to the creation of a severe but reliable and realistic acute hemorrhagic shock model in rats. In the present experiment, rats reliably survived a hemorrhagic shock-induced decrease in mean arterial pressure. Conclusion: The results of this study have shown that the effect of hemorrhage on the decrease of mean arterial blood pres- sure, central vain pressure, blood acidity, and base ex- cess is the same in the presence or absence of soft tissue injury. 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