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Superoxide dismutase activity and malondialdehyde level in plasma and morphological evaluation of acute severe hemorrhagic shock in rats
American Journal of Emergency Medicine (2008) 26, 54 – 58
www.elsevier.com/locate/ajem
Brief Report
Superoxide dismutase activity and malondialdehyde level in plasma and morphological evaluation of acute severe hemorrhagic shock in ratsB Wei Zheng MD, Ling-Zhi Huang, Lian Zhao MD, Bo Wang MD, Hui-Bin Xu MD, Guang-Yi Wang, Zi-Ling Wang PhD, Hong Zhou PhD* Department of Immunohematology, Beijing Institute of Transfusion Medicine, Beijing 100850, China Received 20 November 2006; revised 26 January 2007; accepted 4 February 2007
Abstract Objectives: The aim of the study was to investigate the changes of the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in plasma and organ damage during the acute severe hemorrhagic shock (ASHS), as well as to analyze their relationship. Methods: Twenty male Wistar rats (230-270 g) were randomly divided into sham hemorrhage shock (SHS) group and ASHS group. Acute severe hemorrhagic shock rats were induced by drawing blood through a femoral arterial catheter for 15 minutes with the final mean arterial blood pressure decreased to 35 to 40 mm Hg. The animals were killed after the mean arterial blood pressure was maintained at this level for 1 hour. The activity of SOD and the level of MDA in plasma were measured, and pathologic changes of the major organs (heart, liver, spleen, lung, kidney, and brain) were observed by microscopy. Results: The SOD activities and MDA levels in the ASHS group both increased continuously during the whole experiment. The SOD activities and MDA levels in plasma were not significantly different between the prehemorrhagic stage of ASHS and that of SHS ( P N .05). The SOD activities and MDA levels were significantly higher in the ASHS initial stage than in the prehemorrhagic stage ( P b .01). Compared with the ASHS initial stage, there was a significant ( P b .01) increase in SOD activities and MDA levels in the ASHS end stage. Severe microscopic injuries appeared in the major organs (heart, liver, spleen, lung, kidney, and brain) of ASHS rats. Conclusion: The changes of the activity of SOD and the level of MDA in ASHS may have a positive correlation. D 2008 Elsevier Inc. All rights reserved.
1. Introduction B
This research was supported by grant Y0905001040131 from the science committee of Beijing in China. * Corresponding author. Tel.: +86 10 66931951. E-mail addresses: [email protected] (W. Zheng)8 [email protected] (H. Zhou). 0735-6757/$ – see front matter D 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ajem.2007.02.007
Hemorrhagic shock is 1 of 4 major categories of the syndrome of shock based on cardiovascular characteristics, which was initially proposed in 1972 by Hinshaw and Cox [1]. Hemorrhagic shock is characterized by a loss in circulatory volume, which results in decreased venous return,
SOD activity and MDA level and morphological evaluation of acute severe hemorrhagic shock decreased filling of the cardiac chambers, and hence a decreased cardiac output leading to an increase in the systemic vascular resistance. The hemodynamic profile on monitoring of flow pressure variables shows low central venous pressure, low pulmonary capillary wedge pressure, low cardiac output and cardiac index, and high systemic vascular resistance. The arterial blood pressure may be normal or low [2]. The syndrome of hemorrhagic shock in humans leads to cardiovascular failure and death by impairing the several important organs including heart, liver, spleen, lung, kidney, and brain. It is one of the most common causes of death all over the world today [3] and is paid more and more attention by physicians. Hemorrhagic shock severely and extensively alters the homeostatic mechanisms of the body and elicits complex responses at the organ, cellular, and subcellular levels [4,5]. One of the complicated pathophysiologic manifestations is that free radicals are generated from hypoxia after blood loss. The increased productions of free radicals and other chemical species have been demonstrated in both ischemic and hemorrhagic shock, and oxidative stress is proposed as a fundamental mechanism of organismic damage in these conditions [6,7]. Because free radicals play such an important role in the disease progression, and the radical scavengers or the antioxidant drugs available do not satisfy the clinical treatment, it is necessary for us to profoundly and thoroughly understand the physiologic and pathologic nature of free radicals to disclose a new phenomenon or mechanism from which future clinical tests could benefit. Superoxide dismutase (SOD), acting in the first lines of defense against oxygen-derived free radicals, catalyzes the dismutation of the superoxide anion (O2) into hydrogen peroxide (H2O2), which can be transformed into water and oxygen by catalase. The activity of SOD reflects the cellular capability of scavenging/quenching free radicals [8-10]. Malondialdehyde (MDA), the degradation product of the oxygen-derived free radicals and lipid oxidation, can interfere with the metabolism of protein, glucose, and nucleic acid, which results in the decrease in activity of enzyme, template dysfunction of nucleic acid, and injury of tissues and cells [11]. Then the level of MDA is regarded as the degree of lipid peroxidation. In view of evaluating the basal metabolism of free radicals, the activity of SOD and the level of MDA are the principal pathophysiologic parameters. The current various studies on shock and antioxidant system showed that the changes of the activity of SOD and the level of MDA were always negatively correlated. Decreased SOD activity displays that the cellular antioxygen ability is attenuated, whereas the increased MDA level indicates that cellular injury is aggravated. Moreover, the data reported in the literature on the activity of SOD and the level of MDA were usually focused on reperfusion resuscitation from hemorrhagic shock. Our study is aimed at investigating the changes of the activity of SOD and the
55
level of MDA in plasma and organ damage during acute severe hemorrhagic shock (ASHS), as well as at analyzing their relationship.
2. Materials and methods 2.1. Animals and reagents Male Wistar rats (230-270 g, n = 10 per group) were divided into 2 groups: the sham hemorrhage shock (SHS) rats, which underwent the surgical procedure only, and the ASHS rats. The experiment began after a 24-hour fast. All experiments were approved by our institutional ethical committee for animal welfare. The heparinized saline used during the surgical procedure consisted of 12.5 IU heparin sodium (Peking Chemical Agent Co, Peking, China) per milliliter of normal saline. Pentobarbital sodium (2%, wt/vol) (Peking Chemical Agent Co, Peking, China) was used to anesthetize the rats. Sodium chloride, neutral formalin, alcohol, toluene, hematoxylin, and eosin were purchased from Peking Chemical Agent Co. Superoxide dismutase activity and MDA level test kits were purchased from Nanjing Jiancheng Bioengineering Institute in China.
2.2. Animal preparation The rats were anesthetized during the whole experiment with pentobarbital sodium (2%, wt/vol) by intraperitoneal injection of 0.3 mL per 100 g of body weight. The right carotid artery, the femoral artery, and vein were surgically cannulated with polyethylene catheters asepticly. The blood pressure, rectal temperature, and heart rate were monitored continuously by connecting the arterial catheter to a pressure transducer and computerized physiograph system (Buxco Electronic, Sharon, Conn).
2.3. Acute severe hemorrhagic shock After stabilizing the mean arterial blood pressure (MABP) for 15 minutes, ASHS rats were induced by withdrawing blood through the femoral arterial catheter for 15 minutes. The final MABP decreased to 35 to 40 mm Hg. The MABP was maintained at this level for 1 hour. The total amount of blood withdrawn was then recorded.
2.4. Blood collection The animals were killed 1 hour after ASHS. Blood samples (3 mL) were then collected for the ASHS group (n = 10 for the 3 time intervals: prehemorrhagic stage [0 minute]; ASHS initial stage [15 minutes]; ASHS final stage [75 minutes]). Blood sample (1 mL) was collected for the SHS group (n = 10). The blood samples were centrifuged at 2400g for 10 minutes to obtain plasma. Aliquots were then prepared and stored at 708C until the time of biochemical analysis.
56
W. Zheng et al.
2.5. Biochemical analysis
3.2. Morphological evaluation
The activity of SOD and the level of MDA in plasma were measured by using the SOD test kit and the MDA test kit, respectively.
Severe microscopic injuries appeared in various organs of ASHS rats. There were evidences of acidophilic granular degeneration in some hepatic cells, central veins venectasia, and hyperemia in the liver (Fig. 1a). Telangiectasis and hyperemia were seen in the spatium intermusculare of the heart (Fig. 1b) and in the alveolar septum (Fig. 1c). Several numbers of hyperchromatic and shrunken cortical neurons were observed in the brain (Fig. 1d). There were evidence of leakage of blood and infiltration of lymphocytes into extracellular spaces of the spleen (Fig. 1e) and the kidney (Fig. 1f).
2.6. Histology The animals were killed 1 hour after ASHS. Perfusion was performed with 0.9% sodium chloride solution until there was no blood in the major organs (heart, liver, spleen, lung, kidney, and brain) and then followed by 10% neutral formalin. After the perfusion, the major organs (heart, liver, spleen, lung, kidney, and brain) were dissected and kept overnight at 48C in a similar fixative. The tissues were then dehydrated in an ascending series of alcohol, cleared with toluene, and embedded in paraffin wax. These samples were stained with hematoxylin and eosin and were observed under the light microscope (Olympus, Tokyo, Japan).
2.7. Statistical analysis The results are expressed as mean F SEM. Statistical analysis was undertaken using a 1-way analysis of variance with post hoc least significant difference test. Probability values of less than .05 were considered significant. Statistical analysis was performed using the Statistical Package for Social Science for Windows (SPSSWIN, Chicago, Ill).
3. Results 3.1. The activity of SOD and the level of MDA in plasma The results obtained in the SHS and ASHS groups are shown in Table 1. As seen from Table 1, SOD activities and MDA levels in plasma were not significantly different between the prehemorrhagic stage (A) of ASHS and that of SHS ( P N .05). However, in the ASHS group, SOD activities and MDA levels both increased continuously. Superoxide dismutase activities and MDA levels were significantly higher in the ASHS initial stage (B) than in the prehemorrhagic stage (A) ( P b .01). The ASHS end stage (C) resulted in a significant ( P b .01) increase in SOD activities and MDA levels when compared with the ASHS initial stage (B).
Table 1
4. Discussion Although some possible mechanisms through which oxidative stress exerts a regulatory role in ischemia or/and ASHS are known [5,12-16], several important questions remain unanswered. It is not clear whether oxidative stress and ASHS result from an increased oxidant production or from a failure of antioxidant systems. The importance of SOD activity and MDA level in plasma in ASHS has still been poorly investigated. This study investigated the SOD activity, MDA level, and the histologic examinations of the major organs (liver, heart, lung, brain, spleen, and kidney) to obtain a comprehensive view of antioxidant mechanisms in the ASHS rats. Such studies may provide clues on how oxidative stress may influence the clinical progression of ASHS. Hemorrhagic shock is one of the severe hypovolemic shocks induced by extensive blood loss or sudden and unexpected depletion of the circulating blood. Hemorrhagic shock is a major independent risk factor of the pathogenesis of trauma-related multiple organ failure and death. The pathogenesis of hemorrhagic shock involves multiple interrelated factors including (a) cellular ischemia, (b) circulating or local inflammatory mediators, and (c) free radical injury. Ineffective perfusion leading to cellular ischemia plays a major role in cellular injury in hemorrhagic shock. Hypoperfusion decreases the delivery of nutrients to the cells, leading to diminished adenosine triphosphate (ATP) production. Essential ATP-dependent intracellular metabolic processes that may be affected
Superoxide dismutase activities and MDA levels in the plasma in the SHS and ASHS groups
Parameters
SHS (n = 10)
ASHS (n = 10) A
SOD (U/mg, protein) MDA (nmol/mg, protein)
106.01 F 4.20 2.14 F 0.43
106.13 F 6.21* 2.20 F 0.41*
B
C y
117.00 F 7.28 2.97 F 0.60y
141.04 F 5.03z 4.33 F 1.35z
Data are expressed as mean F SEM. A indicates prehemorrhagic stage (0 minute); B, ASHS initial stage (15 minutes); C, ASHS end stage (75 minutes). * P N .05, no significant difference between groups. y P b .01, significant increase in SOD activities and MDA levels (B vs A). z P b .01, significant increase in SOD activities and MDA levels (C vs B).
SOD activity and MDA level and morphological evaluation of acute severe hemorrhagic shock
57
Fig. 1 The major organs (liver, heart, lung, brain, spleen, and kidney) of SHS and ASHS rats were stained with hematoxylin and eosin. Hematoxylin and eosin stain of liver (A), heart (B), lung (C), brain (D), spleen (E), and kidney (F) of SHS rats were structurally normal. Leakage of blood or infiltrations of lymphocytes into extracellular spaces of major organs were observed in ASHS rats (liver [a], heart [b], lung [c], brain [d], spleen [e], and kidney [f]). All samples were at 100 magnification except for the brain (d). Magnification for the brain (d) was at 40.
58 include maintenance of transmembrane potential, mitochondrial function, and other energy-dependent enzyme reactions. Liver and kidney are particularly sensitive as intracellular levels of ATP fall and ATP-dependent processes are impaired [2]. This was in line with our findings that showed serious morphological injury in the liver (Fig. 1a) and in the kidney (Fig. 1f) in which ATPdependent processes were damaged. Free radicals are natural metabolic coproducts. They are continuously produced when animal body consumes oxygen, such as respiration and some cell-mediated immune functions. They are also generated through environmental pollutants, cigarette smoke, automobile exhaust, radiation, air pollution, pesticides, trauma, ischemia, and so on [17]. Normally there is a balance between the amount of free radicals generated in the body and the antioxidant defense systems that scavenge/quench these free radicals, preventing them from causing deleterious effects in the body [18]. The antioxidant defense systems in the body can only protect the body when the amount of the free radicals is within the normal physiologic level. However, when this balance is shifted toward free radical burden in the body, it leads to oxidative stress, which may result in tissue injury and subsequent diseases [19]. With hemorrhagic shock progressing, under catalytic action of xanthine oxidase, the availability of oxygen generates superoxide, which is converted to hydrogen peroxide and then further reacts to produce the highly reactive tissue-damaging hydroxyl radicals. These oxidative products interact with certain cell targets resulting in cell lysis and tissue injury. One mechanism underlying the effects of oxidative products is that neutrophils are activated to release neutrophil protease and cause amplification of superoxide generation and further tissue damage [20-22]. The increase in MDA levels and the damages in the major organs (liver [Fig. 1a], heart [Fig. 1b], lung [Fig. 1c], brain [Fig. 1d], spleen [Fig. 1e], and kidney [Fig. 1f]) evaluated by morphological analysis in our study verified this doctrine. The ability of cleaning up reactive oxygen species was reduced with SOD generation decreasing, resulting in the development of lipid peroxidation [12]. Some investigators presumed that this was the reason why MDA levels significantly increased. Our data, however, showed a significant increase in SOD activity and MDA level in ASHS compared with SHS. The stimulus of blood loss is severe and long during ASHS. Accumulation of free radicals leads to an increase in lipid peroxidation. Lipid peroxide was estimated by thiobarbituric acid method using MDA as described by Das et al in 1994 [23]. Therefore, MDA level is significantly increased in ASHS when
W. Zheng et al. compared with SHS. At the same time, antioxidant systems are also strongly triggered by the accumulation of free radicals. As a result, SOD activity is greatly augmented. However, if the production velocity of free radicals is much faster than its scavenge/quench velocity, antioxidant systems may be unable to detoxify high levels of reactive oxygen species. Animals without suitable treatment will die because of various organic function exhaustion. Our research demonstrate that SOD activity and MDA level in plasma are both significantly increased in ASHS compared with SHS. However, it is not known what the relationships between SOD activity, MDA level, and organism state are. Further studies are in progress.
References [1] Hinshaw LB, Cox BG. The fundamental mechanisms of shock. New York7 Plenum Press, 1972. [2] Sethi AK, Sharma P. Indian J Anaesth 2003;47:345 - 59. [3] Rodriguez RM, Rosenthal MH. Etiology & pathophysiology of shock. In: Murray MJ, Coursin DB, Pearl RG, et al, editors. Critical care medicine—perioperative management [in English]. London; 2000. p. 192 - 205. [4] Faist E, Wichmann M, Baue AE. The immune response. In: Mattox KL, Feliciano DV, Moore EE, editors. Trauma. 4th ed. McGraw-Hill Companies, Inc; 2000. p. 1409 - 24. [5] Alam H, Stegalkina S, Rhee P, et al. Resuscitation 2002;54:195 - 206. [6] Alexandrova M, Bochev P, Markova V, et al. J Clin Neurosci 2004;11:501 - 6. [7 Cherubini A, Ruggiero C, Polidori MC, et al. Free Radic Biol Med 2005;39:841 - 52. [8] Lantos J, Temes G, Gobolos L, et al. Med Sci Monit 2001;7:1166 - 70. [9] Ilhan N, Halifeoglu I, Qzercan HI, et al. Cell Biochem Funct 2001;19:207 - 12. [10] Wang P, Zhang Y, Tian YK, et al. J Huazhong Univ Sci Technolog Health Sci 2004;33:485 - 7. [11] Su Y, Chen ZY, Cui F, et al. China J Mod Med 2004;4:37 - 40. [12] Altavilla D, Saitta A, Guarini S, et al. Free Radic Biol Med 2001;30:1055 - 66. [13] Powers KA, Zurawska J, Szaszi K, et al. Surgery 2005;137:66 - 74. [14] Sundar SV, Li YY, Rollwagen FM, et al. Biochem Biophys Res Commun 2005;332:688 - 96. [15] Chung TP, Laramie JM, Province M, et al. Crit Care Med 2002;30:S51 - 7. [16] Depre C, Tomlinson JE, Kudej RK, et al. Proc Natl Acad Sci U S A 2001;98:9336 - 41. [17] Govindarajan R, Vijayakumar M, Pushpangadan P. J Ethnopharmacol 2005;99:165 - 78. [18] Nose K. Biol Pharm Bull 2000;23:897 - 903. [19] Finkel T, Holbrook NJ. Nature 2000;408:239 - 47. [20] Guarini S, Altavilla D, Cainazzo MM, et al. Circulation 2003;107: 1189 - 94. [21] Tracey KJ. Nature 2002;420:853 - 9. [22] Guarini S, Cainazzo MM, Giuliani D, et al. Cardiovasc Res 2004;63:357 - 65. [23] Das IJ, Copeland JF, Bushe HS. Medical Physica 1994;21:1733 - 8.
www.elsevier.com/locate/ajem
Brief Report
Superoxide dismutase activity and malondialdehyde level in plasma and morphological evaluation of acute severe hemorrhagic shock in ratsB Wei Zheng MD, Ling-Zhi Huang, Lian Zhao MD, Bo Wang MD, Hui-Bin Xu MD, Guang-Yi Wang, Zi-Ling Wang PhD, Hong Zhou PhD* Department of Immunohematology, Beijing Institute of Transfusion Medicine, Beijing 100850, China Received 20 November 2006; revised 26 January 2007; accepted 4 February 2007
Abstract Objectives: The aim of the study was to investigate the changes of the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in plasma and organ damage during the acute severe hemorrhagic shock (ASHS), as well as to analyze their relationship. Methods: Twenty male Wistar rats (230-270 g) were randomly divided into sham hemorrhage shock (SHS) group and ASHS group. Acute severe hemorrhagic shock rats were induced by drawing blood through a femoral arterial catheter for 15 minutes with the final mean arterial blood pressure decreased to 35 to 40 mm Hg. The animals were killed after the mean arterial blood pressure was maintained at this level for 1 hour. The activity of SOD and the level of MDA in plasma were measured, and pathologic changes of the major organs (heart, liver, spleen, lung, kidney, and brain) were observed by microscopy. Results: The SOD activities and MDA levels in the ASHS group both increased continuously during the whole experiment. The SOD activities and MDA levels in plasma were not significantly different between the prehemorrhagic stage of ASHS and that of SHS ( P N .05). The SOD activities and MDA levels were significantly higher in the ASHS initial stage than in the prehemorrhagic stage ( P b .01). Compared with the ASHS initial stage, there was a significant ( P b .01) increase in SOD activities and MDA levels in the ASHS end stage. Severe microscopic injuries appeared in the major organs (heart, liver, spleen, lung, kidney, and brain) of ASHS rats. Conclusion: The changes of the activity of SOD and the level of MDA in ASHS may have a positive correlation. D 2008 Elsevier Inc. All rights reserved.
1. Introduction B
This research was supported by grant Y0905001040131 from the science committee of Beijing in China. * Corresponding author. Tel.: +86 10 66931951. E-mail addresses: [email protected] (W. Zheng)8 [email protected] (H. Zhou). 0735-6757/$ – see front matter D 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ajem.2007.02.007
Hemorrhagic shock is 1 of 4 major categories of the syndrome of shock based on cardiovascular characteristics, which was initially proposed in 1972 by Hinshaw and Cox [1]. Hemorrhagic shock is characterized by a loss in circulatory volume, which results in decreased venous return,
SOD activity and MDA level and morphological evaluation of acute severe hemorrhagic shock decreased filling of the cardiac chambers, and hence a decreased cardiac output leading to an increase in the systemic vascular resistance. The hemodynamic profile on monitoring of flow pressure variables shows low central venous pressure, low pulmonary capillary wedge pressure, low cardiac output and cardiac index, and high systemic vascular resistance. The arterial blood pressure may be normal or low [2]. The syndrome of hemorrhagic shock in humans leads to cardiovascular failure and death by impairing the several important organs including heart, liver, spleen, lung, kidney, and brain. It is one of the most common causes of death all over the world today [3] and is paid more and more attention by physicians. Hemorrhagic shock severely and extensively alters the homeostatic mechanisms of the body and elicits complex responses at the organ, cellular, and subcellular levels [4,5]. One of the complicated pathophysiologic manifestations is that free radicals are generated from hypoxia after blood loss. The increased productions of free radicals and other chemical species have been demonstrated in both ischemic and hemorrhagic shock, and oxidative stress is proposed as a fundamental mechanism of organismic damage in these conditions [6,7]. Because free radicals play such an important role in the disease progression, and the radical scavengers or the antioxidant drugs available do not satisfy the clinical treatment, it is necessary for us to profoundly and thoroughly understand the physiologic and pathologic nature of free radicals to disclose a new phenomenon or mechanism from which future clinical tests could benefit. Superoxide dismutase (SOD), acting in the first lines of defense against oxygen-derived free radicals, catalyzes the dismutation of the superoxide anion (O2) into hydrogen peroxide (H2O2), which can be transformed into water and oxygen by catalase. The activity of SOD reflects the cellular capability of scavenging/quenching free radicals [8-10]. Malondialdehyde (MDA), the degradation product of the oxygen-derived free radicals and lipid oxidation, can interfere with the metabolism of protein, glucose, and nucleic acid, which results in the decrease in activity of enzyme, template dysfunction of nucleic acid, and injury of tissues and cells [11]. Then the level of MDA is regarded as the degree of lipid peroxidation. In view of evaluating the basal metabolism of free radicals, the activity of SOD and the level of MDA are the principal pathophysiologic parameters. The current various studies on shock and antioxidant system showed that the changes of the activity of SOD and the level of MDA were always negatively correlated. Decreased SOD activity displays that the cellular antioxygen ability is attenuated, whereas the increased MDA level indicates that cellular injury is aggravated. Moreover, the data reported in the literature on the activity of SOD and the level of MDA were usually focused on reperfusion resuscitation from hemorrhagic shock. Our study is aimed at investigating the changes of the activity of SOD and the
55
level of MDA in plasma and organ damage during acute severe hemorrhagic shock (ASHS), as well as at analyzing their relationship.
2. Materials and methods 2.1. Animals and reagents Male Wistar rats (230-270 g, n = 10 per group) were divided into 2 groups: the sham hemorrhage shock (SHS) rats, which underwent the surgical procedure only, and the ASHS rats. The experiment began after a 24-hour fast. All experiments were approved by our institutional ethical committee for animal welfare. The heparinized saline used during the surgical procedure consisted of 12.5 IU heparin sodium (Peking Chemical Agent Co, Peking, China) per milliliter of normal saline. Pentobarbital sodium (2%, wt/vol) (Peking Chemical Agent Co, Peking, China) was used to anesthetize the rats. Sodium chloride, neutral formalin, alcohol, toluene, hematoxylin, and eosin were purchased from Peking Chemical Agent Co. Superoxide dismutase activity and MDA level test kits were purchased from Nanjing Jiancheng Bioengineering Institute in China.
2.2. Animal preparation The rats were anesthetized during the whole experiment with pentobarbital sodium (2%, wt/vol) by intraperitoneal injection of 0.3 mL per 100 g of body weight. The right carotid artery, the femoral artery, and vein were surgically cannulated with polyethylene catheters asepticly. The blood pressure, rectal temperature, and heart rate were monitored continuously by connecting the arterial catheter to a pressure transducer and computerized physiograph system (Buxco Electronic, Sharon, Conn).
2.3. Acute severe hemorrhagic shock After stabilizing the mean arterial blood pressure (MABP) for 15 minutes, ASHS rats were induced by withdrawing blood through the femoral arterial catheter for 15 minutes. The final MABP decreased to 35 to 40 mm Hg. The MABP was maintained at this level for 1 hour. The total amount of blood withdrawn was then recorded.
2.4. Blood collection The animals were killed 1 hour after ASHS. Blood samples (3 mL) were then collected for the ASHS group (n = 10 for the 3 time intervals: prehemorrhagic stage [0 minute]; ASHS initial stage [15 minutes]; ASHS final stage [75 minutes]). Blood sample (1 mL) was collected for the SHS group (n = 10). The blood samples were centrifuged at 2400g for 10 minutes to obtain plasma. Aliquots were then prepared and stored at 708C until the time of biochemical analysis.
56
W. Zheng et al.
2.5. Biochemical analysis
3.2. Morphological evaluation
The activity of SOD and the level of MDA in plasma were measured by using the SOD test kit and the MDA test kit, respectively.
Severe microscopic injuries appeared in various organs of ASHS rats. There were evidences of acidophilic granular degeneration in some hepatic cells, central veins venectasia, and hyperemia in the liver (Fig. 1a). Telangiectasis and hyperemia were seen in the spatium intermusculare of the heart (Fig. 1b) and in the alveolar septum (Fig. 1c). Several numbers of hyperchromatic and shrunken cortical neurons were observed in the brain (Fig. 1d). There were evidence of leakage of blood and infiltration of lymphocytes into extracellular spaces of the spleen (Fig. 1e) and the kidney (Fig. 1f).
2.6. Histology The animals were killed 1 hour after ASHS. Perfusion was performed with 0.9% sodium chloride solution until there was no blood in the major organs (heart, liver, spleen, lung, kidney, and brain) and then followed by 10% neutral formalin. After the perfusion, the major organs (heart, liver, spleen, lung, kidney, and brain) were dissected and kept overnight at 48C in a similar fixative. The tissues were then dehydrated in an ascending series of alcohol, cleared with toluene, and embedded in paraffin wax. These samples were stained with hematoxylin and eosin and were observed under the light microscope (Olympus, Tokyo, Japan).
2.7. Statistical analysis The results are expressed as mean F SEM. Statistical analysis was undertaken using a 1-way analysis of variance with post hoc least significant difference test. Probability values of less than .05 were considered significant. Statistical analysis was performed using the Statistical Package for Social Science for Windows (SPSSWIN, Chicago, Ill).
3. Results 3.1. The activity of SOD and the level of MDA in plasma The results obtained in the SHS and ASHS groups are shown in Table 1. As seen from Table 1, SOD activities and MDA levels in plasma were not significantly different between the prehemorrhagic stage (A) of ASHS and that of SHS ( P N .05). However, in the ASHS group, SOD activities and MDA levels both increased continuously. Superoxide dismutase activities and MDA levels were significantly higher in the ASHS initial stage (B) than in the prehemorrhagic stage (A) ( P b .01). The ASHS end stage (C) resulted in a significant ( P b .01) increase in SOD activities and MDA levels when compared with the ASHS initial stage (B).
Table 1
4. Discussion Although some possible mechanisms through which oxidative stress exerts a regulatory role in ischemia or/and ASHS are known [5,12-16], several important questions remain unanswered. It is not clear whether oxidative stress and ASHS result from an increased oxidant production or from a failure of antioxidant systems. The importance of SOD activity and MDA level in plasma in ASHS has still been poorly investigated. This study investigated the SOD activity, MDA level, and the histologic examinations of the major organs (liver, heart, lung, brain, spleen, and kidney) to obtain a comprehensive view of antioxidant mechanisms in the ASHS rats. Such studies may provide clues on how oxidative stress may influence the clinical progression of ASHS. Hemorrhagic shock is one of the severe hypovolemic shocks induced by extensive blood loss or sudden and unexpected depletion of the circulating blood. Hemorrhagic shock is a major independent risk factor of the pathogenesis of trauma-related multiple organ failure and death. The pathogenesis of hemorrhagic shock involves multiple interrelated factors including (a) cellular ischemia, (b) circulating or local inflammatory mediators, and (c) free radical injury. Ineffective perfusion leading to cellular ischemia plays a major role in cellular injury in hemorrhagic shock. Hypoperfusion decreases the delivery of nutrients to the cells, leading to diminished adenosine triphosphate (ATP) production. Essential ATP-dependent intracellular metabolic processes that may be affected
Superoxide dismutase activities and MDA levels in the plasma in the SHS and ASHS groups
Parameters
SHS (n = 10)
ASHS (n = 10) A
SOD (U/mg, protein) MDA (nmol/mg, protein)
106.01 F 4.20 2.14 F 0.43
106.13 F 6.21* 2.20 F 0.41*
B
C y
117.00 F 7.28 2.97 F 0.60y
141.04 F 5.03z 4.33 F 1.35z
Data are expressed as mean F SEM. A indicates prehemorrhagic stage (0 minute); B, ASHS initial stage (15 minutes); C, ASHS end stage (75 minutes). * P N .05, no significant difference between groups. y P b .01, significant increase in SOD activities and MDA levels (B vs A). z P b .01, significant increase in SOD activities and MDA levels (C vs B).
SOD activity and MDA level and morphological evaluation of acute severe hemorrhagic shock
57
Fig. 1 The major organs (liver, heart, lung, brain, spleen, and kidney) of SHS and ASHS rats were stained with hematoxylin and eosin. Hematoxylin and eosin stain of liver (A), heart (B), lung (C), brain (D), spleen (E), and kidney (F) of SHS rats were structurally normal. Leakage of blood or infiltrations of lymphocytes into extracellular spaces of major organs were observed in ASHS rats (liver [a], heart [b], lung [c], brain [d], spleen [e], and kidney [f]). All samples were at 100 magnification except for the brain (d). Magnification for the brain (d) was at 40.
58 include maintenance of transmembrane potential, mitochondrial function, and other energy-dependent enzyme reactions. Liver and kidney are particularly sensitive as intracellular levels of ATP fall and ATP-dependent processes are impaired [2]. This was in line with our findings that showed serious morphological injury in the liver (Fig. 1a) and in the kidney (Fig. 1f) in which ATPdependent processes were damaged. Free radicals are natural metabolic coproducts. They are continuously produced when animal body consumes oxygen, such as respiration and some cell-mediated immune functions. They are also generated through environmental pollutants, cigarette smoke, automobile exhaust, radiation, air pollution, pesticides, trauma, ischemia, and so on [17]. Normally there is a balance between the amount of free radicals generated in the body and the antioxidant defense systems that scavenge/quench these free radicals, preventing them from causing deleterious effects in the body [18]. The antioxidant defense systems in the body can only protect the body when the amount of the free radicals is within the normal physiologic level. However, when this balance is shifted toward free radical burden in the body, it leads to oxidative stress, which may result in tissue injury and subsequent diseases [19]. With hemorrhagic shock progressing, under catalytic action of xanthine oxidase, the availability of oxygen generates superoxide, which is converted to hydrogen peroxide and then further reacts to produce the highly reactive tissue-damaging hydroxyl radicals. These oxidative products interact with certain cell targets resulting in cell lysis and tissue injury. One mechanism underlying the effects of oxidative products is that neutrophils are activated to release neutrophil protease and cause amplification of superoxide generation and further tissue damage [20-22]. The increase in MDA levels and the damages in the major organs (liver [Fig. 1a], heart [Fig. 1b], lung [Fig. 1c], brain [Fig. 1d], spleen [Fig. 1e], and kidney [Fig. 1f]) evaluated by morphological analysis in our study verified this doctrine. The ability of cleaning up reactive oxygen species was reduced with SOD generation decreasing, resulting in the development of lipid peroxidation [12]. Some investigators presumed that this was the reason why MDA levels significantly increased. Our data, however, showed a significant increase in SOD activity and MDA level in ASHS compared with SHS. The stimulus of blood loss is severe and long during ASHS. Accumulation of free radicals leads to an increase in lipid peroxidation. Lipid peroxide was estimated by thiobarbituric acid method using MDA as described by Das et al in 1994 [23]. Therefore, MDA level is significantly increased in ASHS when
W. Zheng et al. compared with SHS. At the same time, antioxidant systems are also strongly triggered by the accumulation of free radicals. As a result, SOD activity is greatly augmented. However, if the production velocity of free radicals is much faster than its scavenge/quench velocity, antioxidant systems may be unable to detoxify high levels of reactive oxygen species. Animals without suitable treatment will die because of various organic function exhaustion. Our research demonstrate that SOD activity and MDA level in plasma are both significantly increased in ASHS compared with SHS. However, it is not known what the relationships between SOD activity, MDA level, and organism state are. Further studies are in progress.
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