Burn Injury Induces Skeletal Muscle Degeneration, Inflammatory Host Response, and Oxidative Stress in Wistar Rats Nathalia Trasmonte da Silva, Hananiah Tardivo Quintana, Jeferson André Bortolin, Daniel Araki Ribeiro, PhD, Flavia de Oliveira, PhD
Burn injuries (BIs) result in both local and systemic responses distant from the site of thermal injury, such as skeletal muscle. The purpose of this study was to investigate the expression of cyclooxygenase-2 (COX-2) and hydroxy-2′-deoxyguanosine (8-OHdG) as a result of inflammation and reactive oxygen species production, respectively. A total of 16 male rats were distributed into two groups: control (C) and submitted to BI. The medial part of gastrocnemius muscle formed the specimens, which were stained with hematoxylin and eosin and were evaluated. COX-2 and 8-OHdG expressions were assessed by immunohistochemistry, and cell profile area and density of muscle fibers (number of fibers per square millimeter) were evaluated by morphometric methods. The results revealed inflammatory infiltrate associated with COX-2 immunoexpression in BI-gastrocnemius muscle. Furthermore, a substantial decrease in the muscle cell profile area of BI group was noticed when compared with the control group, whereas the density of muscle fibers was higher in the BI group. 8-OHdG expression in numerous skeletal muscle nuclei was detected in the BI group. In conclusion, the BI group is able to induce skeletal muscle degeneration as a result of systemic host response closely related to reactive oxygen species production and inflammatory process. (J Burn Care Res 2015;36:428–433)
Burn injury (BI) has been associated with protein catabolism characterized by a hypermetabolic response. Patients with burns greater than 40% total surface area have a catabolic response.1 This will affect their metabolism and persist at least 1 year after injury in most body tissues,2 and most of the burns occur in children of age under 5 years,3 when the organs are developing. These BIs result in both local and systemic responses distant from the site of thermal injury, such as skeletal muscle,4–7 as a result of systemic host response. After a BI, there is an enormous production of reactive oxygen species (ROS), which is harmful and implicated in inflammation, systemic inflammatory
From the Department of Biosciences, Federal University of São Paulo (UNIFESP), Santos, Brazil. Supported by FAPESP (process number 11/22034-9). Address correspondence to Flavia de Oliveira, Department of Biosciences, Federal University of São Paulo (UNIFESP), Rua Silva Jardim, 136 - Lab 328, Santos, São Paulo 11015-020, Brazil. Email: [email protected]. Copyright © 2015 by the American Burn Association 1559-047X/2015 DOI: 10.1097/BCR.0000000000000122
428
response syndrome, immunosuppression, infection and sepsis, tissue damage, and multiple organ failure.8 According to some authors, oxidative damage is one of the mechanisms responsible for the local and distant pathophysiological events observed after pathophysiologic process. One among the inflammatory mediators associated with muscle repair that are described here is cyclooxygenase-2 (COX-2). It is an enzyme responsible for the conversion of arachidonic acid to prostaglandins9 and is very important in many pathological processes, including inflammation.10 Inflammatory cells also produce ROS. Several studies have demonstrated that burn initiates systemic inflammatory reactions by producing ROS, which leads to lipid peroxidation.7,11–13 Recent evidences suggest that overproduction of ROS can result in DNA fragmentation, lipid peroxidation, and protein oxidation, which can lead to apoptosis.14 Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the predominant forms of free radical that cause oxidative stress, and therefore, it has been widely used as a putative biomarker for oxidative stress.15 According to Xie et al,16 8-OHdG is a suitable biological marker of
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oxidative cellular damage. ROS and reactive nitrogen species release 8-hydroxylation of guanine base, being the damaged DNA is repaired in vivo by endonucleases. Therefore, the purpose of this study was to investigate the expression of COX-2 and 8-OHdG in a skeletal muscle distant from BI as a result of inflammation and ROS production, respectively. Certainly, such data will contribute to a better understanding of biological mechanisms involved in BI.
METHODS Animals and Experimental Design A total of sixteen 21-day-old male Wistar rats (Rattus norvegicus) were chosen in this study to mimic a developing organism. The rats were individually housed in cages for 5 days in a temperature-controlled room (21–24°C) with a regular light–dark cycle. On the sixth day, the animals were anesthetized with an intraperitoneal injection of ketamine (50 mg/kg) and xilazyne (10 mg/kg), and the dorsal and ventral hair was removed. A nonlethal scald injury on the skin was administered to the eight animals constituting the BI group by immersing 45% of each rat’s body in 87°C water, as described by Walker and Mason.17 Each animal had 30% of its dorsal and 15% of its ventral area exposed to scald injury for 10 and 3 seconds, respectively.18 The rats in both groups were subcutaneously injected with the analgesic buprenorphine (0.2 mg/ kg) immediately after BI and 24 hours later. After 14 days following the scald injury, all animals from each group were euthanized with a lethal intraperitoneal injection of ketamine (150 mg/kg) and xilazyne (30 mg/kg). All procedures were approved by the Committee of Ethics and Research from the Federal University of São Paulo (protocol number 158/12).
Histopathological Analysis The gastrocnemius muscles of rats from the BI and control (C) groups were examined. The medial part of each gastrocnemius was dissected and transversely sectioned into proximal, middle, and distal parts. The middle part was immediately fixed in 10% formalin phosphate buffer for 24 hours for histological analyzes. The specimens were routinely embedded in paraffin blocks and cut in transverse sections (5 μm). The slides were stained with hematoxylin and eosin (H&E).
Morphometric Study: Cell Profile Area and Density of Muscle Fibers The average profile of muscle fibers was determined from the measurement of 30 muscle fibers stained
da Silva et al 429
with H&E. These fibers were randomly chosen from each animal comprising each experimental group. To determine the fiber area in square millimeter, we used computerized imaging equipment (Axio Visio, Zeiss®, Carl Zeiss, Oberkochen, Germany) attached to a binocular microscope (Axio Observer D1, Zeiss®) with a ×40 objective. The total density of muscle fibers (number of fibers per square millimeter) was determined as described by Mandarim-de-Lacerda.19 For this purpose, two cuts chosen randomly from the total of 10 sections stained with H&E were used. In each section, three photomicrographs were obtained with the aid of computerized imaging system (Axio Visio, Zeiss®) attached to a binocular light microscope (Axio Observer D1, Zeiss®). A total of six photomicrographs were assessed per animal.
Immunohistochemistry Analysis Slides with sections were pretreated in a microwave with 0.01 M citric acid buffer (pH 6) for three cycles of 5 minutes each at 850 W for antigen retrieval. The material was preincubated with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) solution for 5 minutes to inactivate the endogenous peroxidase and then blocked with 5% normal goat serum in PBS solution for 10 minutes. The specimens were then incubated with anti-COX-2 polyclonal primary antibody (SC-1747, Santa Cruz Biotechnology, Dallas, TX) at a concentration of 1:200 and with 8-OHdG (SC66036, Santa Cruz Biotechnology) at 1:100 concentration. Incubations were performed overnight at 4°C in a refrigerator. This was followed by two washes in PBS for 10 minutes. The sections were then incubated with biotin-conjugated secondary antibody anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at a concentration of 1:200 in PBS for 1 hour. The sections were washed twice with PBS followed by the application of performed avidin–biotin complex conjugated to peroxidase (Vector Laboratories) for 45 minutes. The bound complexes were visualized by the application of a 0.05% solution of 3,3′-diaminobenzidine solution and counterstained with Harris hematoxylin. For control studies of the antibodies, the serial sections were treated with rabbit IgG (Vector Laboratories) at a concentration of 1:200 in place of the primary antibody.
Statistical Treatment The statistical method used for determining the profile area of the muscle fibers and the density of these fibers was the Student’s t-test for unrelated samples. P < .05 was considered to be statistically significant.
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Figure 1. Histopathological evaluation of rat gastrocnemius muscle stained with hematoxylin and eosin. A. Control (C) group. B–D. Burn injury (BI) group. In (B–D), big arrows indicate inflammatory cells coming from vessels transversely sectioned (asterisk) with vascular congestion. Note inflammatory cells intermuscle fibers (small arrows) in (B) and (C). Bar = 50 μm.
RESULTS Histopathological Analysis The histopathological analysis revealed normal fibers distributed in muscle fascicles, which were identified equidistantly in the control group (Figure 1A). By contrast, the BI group revealed histopathological changes, such as inflammatory cells between muscles fascicles (Figure 1B–D, big arrows) and inflammatory infiltrate between muscle fibers (Figure 1B, C, small arrows). Vascular congestion in the BI group was also noted (Figure 1B–D, asterisk).
quantified data presented in Figures 2 and 3 were derived from H&E-stained sections.
Immunohistochemistry Data COX-2 expression was detected predominantly in the cytoplasm of muscle cells. A higher immunoreactivity was detected in the BI group (Figure 4B) when compared with the normal counterpart (Figure 4A).
Morphometric Results Morphometric data pointed out statistically significant differences in cell profile area and density of muscle fibers (number of fibers per square millimeter). The cell profile area of the BI group was decreased when compared with that of the control group (P < .05). However, the density of the muscle fibers in the BI group was increased when compared with that in the control group (P < .05). All
Figure 2. Mean and stand deviation of cell profile area. C represents control group and BI, burn injury group. *P < .05.
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Figure 3. Mean and stand deviation of density of muscle fibers (number of fibers per square millimeter). C represents control group and BI, burn injury group. *P < .05.
8-OHdG immunoexpression was detected in the nucleus of muscle cells. In the control group, immunoexpression was not detected (Figure 5A). For this reason, the immunoreactivitiy was considered negative in the control group. Regarding the BI group, a strong 8-OHdG expression was detected (Figure 5B).
DISCUSSION The purpose of this study was to investigate the immunoexpresssion of COX-2 and 8-OHdG in rats subjected to BI in a muscle distant from injury as a result of inflammatory host response and oxidative stress, respectively. The approach has not been demonstrated so far. In this experimental model of BI, it was possible to characterize inflammation signs in a skeletal muscle distant from injury. Herein, histopathological evaluation showed the presence of inflammatory infiltrate between muscle fibers and vascular blood congestion in the vessels of burn-injured animals.
da Silva et al 431
Morphometric data revealed statistically significant differences in cell profile area and density of muscle fibers (number of fibers per square millimeter). In particular, the cell profile area of the BI group was decreased when compared with that of the control group, whereas the density of the muscle fibers in the BI group was increased when compared with that in the control group. These findings are new and therefore are quite complicated to discuss. It seems that BI is able to induce skeletal muscle degeneration as a result of muscle atrophy by means of decreased cell profile area followed by compensatory muscle fiber regeneration. Further studies are needed to elucidate the issue. To understand the role of inflammatory host response, we investigated the COX-2 expression in skeletal-muscle cells after BI. It is well established that muscle regeneration is triggered by a number of growth factors and cytokines. However, many biological phenomena closely related to regulation of muscle regeneration remain unclear and most likely involve molecules that have to be defined yet.20 Anyway, several studies have demonstrated that COX-2 plays a crucial role after skeletal-muscle injury, using different experimental models.20–22 Nevertheless, studies investigating COX-2 expression in muscle distant from BI and as a consequence of systemic inflammatory response were not performed so far. Our results revealed that COX-2 had a strong immunoexpresssion in the BI group when compared with the control group. According to Bondesen et al,23 COX-2 expression in skeletal muscle is related to myofibrillar muscle regeneration, that is, COX-2 activity is important for muscle growth under distinct biological pathways. These data suggest that COX-2 is required during early stages of muscle
Figure 4. Cyclooxygenase-2 (COX-2) immunoexpression in gastrocnemius muscle. A. Control group. B. Burn injury group. Bar = 100 μm.
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Figure 5. Hydroxy-2′-deoxyguanosine (8-OHdG) immunoexpression in gastrocnemius muscle. A. Control group. B. Burn injury group. Arrows show immunoreactive nuclei. Bar = 100 μm.
regeneration.20 The mechanism by which the number of myoblasts in regenerating muscle is decreased by the lack of COX-2 activity may be either extrinsic or intrinsic pathways. The most likely muscleextrinsic function of COX-2 is in the inflammatory response after muscle injury. Muscle regeneration is impaired when inflammatory cells are depleted and stimulated when they are increased,24 demonstrating the importance of inflammation after muscle damage. Neutrophils and macrophages that infiltrate damaged muscle not only promote phagocytosis process but also secrete growth factors and chemoattractants, such as COX-2 that is implicated in myogenesis.25 Thus, evidence suggests that muscle regeneration involves complex interactions between inflammatory and muscle cells. Such findings are in agreement with our results. Inflammatory cells produce ROS, which are synthesized mainly during oxidative phosphorylation and activation of phagocytic cells during oxidative explosion.8,13 Excessive production of ROS can lead to damage to lipids, proteins, membranes, and nucleic acids, and it also serves as an important intracellular marker that amplifies inflammatory responses. To further elucidate the presence of ROS after BI, 8-OHdG immunohistochemistry was performed in this experimental design. It has been established that 8-OHdG damages DNA and can be easily detected in the urine of burn-injured patients,16 but it has never been properly investigated in skeletal muscle in situ as a consequence of BI. Our results demonstrated a strong 8-OHdG expression in many nuclei of muscle cells in the BI group, whereas in the control group, immunoexpression was not detected. Sin et al26 investigated
rats with deep-tissue injury, 8-OHdG immunoreactivity was found to be present in the skeletal muscle. The authors assumed that oxidative stress and DNA damage signaling in skeletal muscle are involved in the underlying mechanisms responsible for the pathogenesis of deep-tissue injury. Taken together, we postulated that BI induces oxidative stress and DNA damage in the skeletal muscle that is distant from the site of origin of thermal injury by means of 8-OHdG immunoexpression. In conclusion, BI is able to induce skeletal-muscle degeneration as a result of systemic host response closely related to ROS production and inflammatory host response of Wistar rats. REFERENCES 1. Porter C, Hurren NM, Herndon DN, Børsheim E. Whole body and skeletal muscle protein turnover in recovery from burns. Int J Burns Trauma 2013;3:9–17. 2. Pereira CT, Murphy KD, Herndon DN. Altering metabolism. J Burn Care Rehabil 2005;26:194–9. 3. Krishnamoorthy V, Ramaiah R, Bhananker SM. Pediatric burn injuries. Int J Crit Illn Inj Sci 2012;2:128–34. 4. Fang CH, Li BG, Fischer DR, et al. Burn injury upregulates the activity and gene expression of the 20 S proteasome in rat skeletal muscle. Clin Sci (Lond) 2000;99:181–7. 5. Hart DW, Wolf SE, Chinkes DL, et al. Determinants of skeletal muscle catabolism after severe burn. Ann Surg 2000;232:455–65. 6. Oliveira Fd, Bevilacqua LR, Anaruma CA, Boldrini Sde C, Liberti EA. Morphological changes in distant muscle fibers following thermal injury in Wistar rats. Acta Cir Bras 2010;25:525–8. 7. Wu X, Wolf SE, Walters TJ. Muscle contractile properties in severely burned rats. Burns 2010;36:905–11. 8. Parihar A, Parihar MS, Milner S, Bhat S. Oxidative stress and anti-oxidative mobilization in burn injury. Burns 2008;34:6–17. 9. Paiotti AP, Marchi P, Miszputen SJ, Oshima CT, Franco M, Ribeiro DA. The role of nonsteroidal antiinflammatory
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10.
11. 12.
13. 14.
15.
16. 17.
drugs and cyclooxygenase-2 inhibitors on experimental colitis. In Vivo 2012;26:381–93. Shibata M, Kodani I, Osaki M, et al. Cyclo-oxygenase-1 and -2 expression in human oral mucosa, dysplasias and squamous cell carcinomas and their pathological significance. Oral Oncol 2005;41:304–12. Horton JW. Free radicals and lipid peroxidation mediated injury in burn trauma: the role of antioxidant therapy. Toxicology 2003;189:75–88. Hoşnuter M, Gürel A, Babucçu O, Armutcu F, Kargi E, Işikdemir A. The effect of CAPE on lipid peroxidation and nitric oxide levels in the plasma of rats following thermal injury. Burns 2004;30:121–5. Xue C, Chou CS, Kao CY, Sen CK, Friedman A. Propagation of cutaneous thermal injury: a mathematical model. Wound Repair Regen 2012;20:114–22. Fujita R, Tanaka Y, Saihara Y, Yamakita M, Ando D, Koyama K. Effect of molecular hydrogen saturated alkaline electrolyzed water on disuse muscle atrophy in gastrocnemius muscle. J Physiol Anthropol 2011;30:195–201. Valavanidis A, Vlachogianni T, Fiotakis C. 8-Hydroxy-2′deoxyguanosine (8-OHdG): a critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 2009;27:120–39. Xie XQ, Shinozawa Y, Sasaki J, et al. Neuroendocrine system response modulates oxidative cellular damage in burn patients. Tohoku J Exp Med 2007;211:161–9. Walker HL, Mason AD Jr. A standard animal burn. J Trauma 1968;8:1049–51.
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18. Newman JJ, Strome DR, Goodwin CW, Mason AD Jr, Pruitt BA Jr. Altered muscle metabolism in rats after thermal injury. Metabolism 1982;31:1229–33. 19. Mandarim-de-Lacerda CA. Stereological tools in biomedical research. Ann Brazil Acad Sci 2003;75:469–486. 20. Bondesen BA, Mills ST, Kegley KM, Pavlath GK. The COX2 pathway is essential during early stages of skeletal muscle regeneration. Am J Physiol Cell Physiol 2004;287:C475–83. 21. Rennó AC, Toma RL, Feitosa SM, et al. Comparative effects of low-intensity pulsed ultrasound and low-level laser therapy on injured skeletal muscle. Photomed Laser Surg 2011;29:5–10. 22. de Oliveira F, Quintana HT, Bortolin JA, et al. Cyclo oxygenase-2 expression in skeletal muscle of knockout mice suffering Duchenne muscular dystrophy. Histochem Cell Biol 2013;139:685–9. 23. Bondesen BA, Mills ST, Pavlath GK. The COX-2 pathway regulates growth of atrophied muscle via multiple mechanisms. Am J Physiol Cell Physiol 2006;290:C1651–9. 24. Lescaudron L, Peltékian E, Fontaine-Pérus J, et al. Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant. Neuromuscul Disord 1999;9:72–80. 25. Cassatella MA. Neutrophil-derived proteins: selling cyto kines by the pound. Adv Immunol 1999;73:369–509. 26. Sin TK, Pei XM, Teng BT, Tam EW, Yung BY, Siu PM. Oxidative stress and DNA damage signalling in skeletal muscle in pressure-induced deep tissue injury. Pflugers Arch 2013;465:295–317.
Burn injuries (BIs) result in both local and systemic responses distant from the site of thermal injury, such as skeletal muscle. The purpose of this study was to investigate the expression of cyclooxygenase-2 (COX-2) and hydroxy-2′-deoxyguanosine (8-OHdG) as a result of inflammation and reactive oxygen species production, respectively. A total of 16 male rats were distributed into two groups: control (C) and submitted to BI. The medial part of gastrocnemius muscle formed the specimens, which were stained with hematoxylin and eosin and were evaluated. COX-2 and 8-OHdG expressions were assessed by immunohistochemistry, and cell profile area and density of muscle fibers (number of fibers per square millimeter) were evaluated by morphometric methods. The results revealed inflammatory infiltrate associated with COX-2 immunoexpression in BI-gastrocnemius muscle. Furthermore, a substantial decrease in the muscle cell profile area of BI group was noticed when compared with the control group, whereas the density of muscle fibers was higher in the BI group. 8-OHdG expression in numerous skeletal muscle nuclei was detected in the BI group. In conclusion, the BI group is able to induce skeletal muscle degeneration as a result of systemic host response closely related to reactive oxygen species production and inflammatory process. (J Burn Care Res 2015;36:428–433)
Burn injury (BI) has been associated with protein catabolism characterized by a hypermetabolic response. Patients with burns greater than 40% total surface area have a catabolic response.1 This will affect their metabolism and persist at least 1 year after injury in most body tissues,2 and most of the burns occur in children of age under 5 years,3 when the organs are developing. These BIs result in both local and systemic responses distant from the site of thermal injury, such as skeletal muscle,4–7 as a result of systemic host response. After a BI, there is an enormous production of reactive oxygen species (ROS), which is harmful and implicated in inflammation, systemic inflammatory
From the Department of Biosciences, Federal University of São Paulo (UNIFESP), Santos, Brazil. Supported by FAPESP (process number 11/22034-9). Address correspondence to Flavia de Oliveira, Department of Biosciences, Federal University of São Paulo (UNIFESP), Rua Silva Jardim, 136 - Lab 328, Santos, São Paulo 11015-020, Brazil. Email: [email protected]. Copyright © 2015 by the American Burn Association 1559-047X/2015 DOI: 10.1097/BCR.0000000000000122
428
response syndrome, immunosuppression, infection and sepsis, tissue damage, and multiple organ failure.8 According to some authors, oxidative damage is one of the mechanisms responsible for the local and distant pathophysiological events observed after pathophysiologic process. One among the inflammatory mediators associated with muscle repair that are described here is cyclooxygenase-2 (COX-2). It is an enzyme responsible for the conversion of arachidonic acid to prostaglandins9 and is very important in many pathological processes, including inflammation.10 Inflammatory cells also produce ROS. Several studies have demonstrated that burn initiates systemic inflammatory reactions by producing ROS, which leads to lipid peroxidation.7,11–13 Recent evidences suggest that overproduction of ROS can result in DNA fragmentation, lipid peroxidation, and protein oxidation, which can lead to apoptosis.14 Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the predominant forms of free radical that cause oxidative stress, and therefore, it has been widely used as a putative biomarker for oxidative stress.15 According to Xie et al,16 8-OHdG is a suitable biological marker of
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oxidative cellular damage. ROS and reactive nitrogen species release 8-hydroxylation of guanine base, being the damaged DNA is repaired in vivo by endonucleases. Therefore, the purpose of this study was to investigate the expression of COX-2 and 8-OHdG in a skeletal muscle distant from BI as a result of inflammation and ROS production, respectively. Certainly, such data will contribute to a better understanding of biological mechanisms involved in BI.
METHODS Animals and Experimental Design A total of sixteen 21-day-old male Wistar rats (Rattus norvegicus) were chosen in this study to mimic a developing organism. The rats were individually housed in cages for 5 days in a temperature-controlled room (21–24°C) with a regular light–dark cycle. On the sixth day, the animals were anesthetized with an intraperitoneal injection of ketamine (50 mg/kg) and xilazyne (10 mg/kg), and the dorsal and ventral hair was removed. A nonlethal scald injury on the skin was administered to the eight animals constituting the BI group by immersing 45% of each rat’s body in 87°C water, as described by Walker and Mason.17 Each animal had 30% of its dorsal and 15% of its ventral area exposed to scald injury for 10 and 3 seconds, respectively.18 The rats in both groups were subcutaneously injected with the analgesic buprenorphine (0.2 mg/ kg) immediately after BI and 24 hours later. After 14 days following the scald injury, all animals from each group were euthanized with a lethal intraperitoneal injection of ketamine (150 mg/kg) and xilazyne (30 mg/kg). All procedures were approved by the Committee of Ethics and Research from the Federal University of São Paulo (protocol number 158/12).
Histopathological Analysis The gastrocnemius muscles of rats from the BI and control (C) groups were examined. The medial part of each gastrocnemius was dissected and transversely sectioned into proximal, middle, and distal parts. The middle part was immediately fixed in 10% formalin phosphate buffer for 24 hours for histological analyzes. The specimens were routinely embedded in paraffin blocks and cut in transverse sections (5 μm). The slides were stained with hematoxylin and eosin (H&E).
Morphometric Study: Cell Profile Area and Density of Muscle Fibers The average profile of muscle fibers was determined from the measurement of 30 muscle fibers stained
da Silva et al 429
with H&E. These fibers were randomly chosen from each animal comprising each experimental group. To determine the fiber area in square millimeter, we used computerized imaging equipment (Axio Visio, Zeiss®, Carl Zeiss, Oberkochen, Germany) attached to a binocular microscope (Axio Observer D1, Zeiss®) with a ×40 objective. The total density of muscle fibers (number of fibers per square millimeter) was determined as described by Mandarim-de-Lacerda.19 For this purpose, two cuts chosen randomly from the total of 10 sections stained with H&E were used. In each section, three photomicrographs were obtained with the aid of computerized imaging system (Axio Visio, Zeiss®) attached to a binocular light microscope (Axio Observer D1, Zeiss®). A total of six photomicrographs were assessed per animal.
Immunohistochemistry Analysis Slides with sections were pretreated in a microwave with 0.01 M citric acid buffer (pH 6) for three cycles of 5 minutes each at 850 W for antigen retrieval. The material was preincubated with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) solution for 5 minutes to inactivate the endogenous peroxidase and then blocked with 5% normal goat serum in PBS solution for 10 minutes. The specimens were then incubated with anti-COX-2 polyclonal primary antibody (SC-1747, Santa Cruz Biotechnology, Dallas, TX) at a concentration of 1:200 and with 8-OHdG (SC66036, Santa Cruz Biotechnology) at 1:100 concentration. Incubations were performed overnight at 4°C in a refrigerator. This was followed by two washes in PBS for 10 minutes. The sections were then incubated with biotin-conjugated secondary antibody anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at a concentration of 1:200 in PBS for 1 hour. The sections were washed twice with PBS followed by the application of performed avidin–biotin complex conjugated to peroxidase (Vector Laboratories) for 45 minutes. The bound complexes were visualized by the application of a 0.05% solution of 3,3′-diaminobenzidine solution and counterstained with Harris hematoxylin. For control studies of the antibodies, the serial sections were treated with rabbit IgG (Vector Laboratories) at a concentration of 1:200 in place of the primary antibody.
Statistical Treatment The statistical method used for determining the profile area of the muscle fibers and the density of these fibers was the Student’s t-test for unrelated samples. P < .05 was considered to be statistically significant.
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Figure 1. Histopathological evaluation of rat gastrocnemius muscle stained with hematoxylin and eosin. A. Control (C) group. B–D. Burn injury (BI) group. In (B–D), big arrows indicate inflammatory cells coming from vessels transversely sectioned (asterisk) with vascular congestion. Note inflammatory cells intermuscle fibers (small arrows) in (B) and (C). Bar = 50 μm.
RESULTS Histopathological Analysis The histopathological analysis revealed normal fibers distributed in muscle fascicles, which were identified equidistantly in the control group (Figure 1A). By contrast, the BI group revealed histopathological changes, such as inflammatory cells between muscles fascicles (Figure 1B–D, big arrows) and inflammatory infiltrate between muscle fibers (Figure 1B, C, small arrows). Vascular congestion in the BI group was also noted (Figure 1B–D, asterisk).
quantified data presented in Figures 2 and 3 were derived from H&E-stained sections.
Immunohistochemistry Data COX-2 expression was detected predominantly in the cytoplasm of muscle cells. A higher immunoreactivity was detected in the BI group (Figure 4B) when compared with the normal counterpart (Figure 4A).
Morphometric Results Morphometric data pointed out statistically significant differences in cell profile area and density of muscle fibers (number of fibers per square millimeter). The cell profile area of the BI group was decreased when compared with that of the control group (P < .05). However, the density of the muscle fibers in the BI group was increased when compared with that in the control group (P < .05). All
Figure 2. Mean and stand deviation of cell profile area. C represents control group and BI, burn injury group. *P < .05.
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Figure 3. Mean and stand deviation of density of muscle fibers (number of fibers per square millimeter). C represents control group and BI, burn injury group. *P < .05.
8-OHdG immunoexpression was detected in the nucleus of muscle cells. In the control group, immunoexpression was not detected (Figure 5A). For this reason, the immunoreactivitiy was considered negative in the control group. Regarding the BI group, a strong 8-OHdG expression was detected (Figure 5B).
DISCUSSION The purpose of this study was to investigate the immunoexpresssion of COX-2 and 8-OHdG in rats subjected to BI in a muscle distant from injury as a result of inflammatory host response and oxidative stress, respectively. The approach has not been demonstrated so far. In this experimental model of BI, it was possible to characterize inflammation signs in a skeletal muscle distant from injury. Herein, histopathological evaluation showed the presence of inflammatory infiltrate between muscle fibers and vascular blood congestion in the vessels of burn-injured animals.
da Silva et al 431
Morphometric data revealed statistically significant differences in cell profile area and density of muscle fibers (number of fibers per square millimeter). In particular, the cell profile area of the BI group was decreased when compared with that of the control group, whereas the density of the muscle fibers in the BI group was increased when compared with that in the control group. These findings are new and therefore are quite complicated to discuss. It seems that BI is able to induce skeletal muscle degeneration as a result of muscle atrophy by means of decreased cell profile area followed by compensatory muscle fiber regeneration. Further studies are needed to elucidate the issue. To understand the role of inflammatory host response, we investigated the COX-2 expression in skeletal-muscle cells after BI. It is well established that muscle regeneration is triggered by a number of growth factors and cytokines. However, many biological phenomena closely related to regulation of muscle regeneration remain unclear and most likely involve molecules that have to be defined yet.20 Anyway, several studies have demonstrated that COX-2 plays a crucial role after skeletal-muscle injury, using different experimental models.20–22 Nevertheless, studies investigating COX-2 expression in muscle distant from BI and as a consequence of systemic inflammatory response were not performed so far. Our results revealed that COX-2 had a strong immunoexpresssion in the BI group when compared with the control group. According to Bondesen et al,23 COX-2 expression in skeletal muscle is related to myofibrillar muscle regeneration, that is, COX-2 activity is important for muscle growth under distinct biological pathways. These data suggest that COX-2 is required during early stages of muscle
Figure 4. Cyclooxygenase-2 (COX-2) immunoexpression in gastrocnemius muscle. A. Control group. B. Burn injury group. Bar = 100 μm.
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Figure 5. Hydroxy-2′-deoxyguanosine (8-OHdG) immunoexpression in gastrocnemius muscle. A. Control group. B. Burn injury group. Arrows show immunoreactive nuclei. Bar = 100 μm.
regeneration.20 The mechanism by which the number of myoblasts in regenerating muscle is decreased by the lack of COX-2 activity may be either extrinsic or intrinsic pathways. The most likely muscleextrinsic function of COX-2 is in the inflammatory response after muscle injury. Muscle regeneration is impaired when inflammatory cells are depleted and stimulated when they are increased,24 demonstrating the importance of inflammation after muscle damage. Neutrophils and macrophages that infiltrate damaged muscle not only promote phagocytosis process but also secrete growth factors and chemoattractants, such as COX-2 that is implicated in myogenesis.25 Thus, evidence suggests that muscle regeneration involves complex interactions between inflammatory and muscle cells. Such findings are in agreement with our results. Inflammatory cells produce ROS, which are synthesized mainly during oxidative phosphorylation and activation of phagocytic cells during oxidative explosion.8,13 Excessive production of ROS can lead to damage to lipids, proteins, membranes, and nucleic acids, and it also serves as an important intracellular marker that amplifies inflammatory responses. To further elucidate the presence of ROS after BI, 8-OHdG immunohistochemistry was performed in this experimental design. It has been established that 8-OHdG damages DNA and can be easily detected in the urine of burn-injured patients,16 but it has never been properly investigated in skeletal muscle in situ as a consequence of BI. Our results demonstrated a strong 8-OHdG expression in many nuclei of muscle cells in the BI group, whereas in the control group, immunoexpression was not detected. Sin et al26 investigated
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