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Heat shock response reduces mortality after severe experimental burns
Burns 26 (2000) 233±238
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Heat shock response reduces mortality after severe experimental burns Tu® Neder Meyer a,*, Alcino LaÂzaro da Silva b, EÃnio Cardillo Vieira b, Antonio Carlos Vassalo Alves b a
INCIS (Health Sciences Institute), UNINCOR (Vale do Rio Verde University), TreÃs Corac° oÄes, MG, Brazil b UFMG (Federal University of Minas Gerais), Belo Horizonte, MG, Brazil Accepted 20 July 1999
Abstract The heat shock response has imparted protective eects in animal models of septic shock and endotoxemia. This study has tested the hypothesis that it could be protective in experimental burns. One hundred and ®fteen adult male Fischer rats were randomly divided into four groups. Rats in the ®rst group (n = 12) were anesthetized and shaved. In the second group (n = 15) rats were anesthetized and heated in a 458C water bath. In the third group (n = 44), rats were anesthetized, shaved and submitted to 26±30% body surface third-degree burns using a brass bar. In the fourth group (n = 44), rats were anesthetized, heated and, 1 day after, they were burnt. Mortality rates were measured at 3, 7, 15 and 25 days. Liver and lung samples were collected from all groups for heat-shock protein 70 detection. Heat-shock protein 70 was positive in heated animals. No animals died in the ®rst or second group. Heated and burnt animals showed signi®cantly decreased mortality at days 3 ( p < 0.05, Fischer's exact test) and at days 7, 15 and 25 ( p < 0.01) after burns, when compared to unheated burnt animals. In conclusion, eliciting the heat-shock response signi®cantly reduced mortality rates in this model of experimental burns. # 2000 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Heat-shock proteins; Stress proteins; Heat-shock response; Stress response; Burns; Survival rate; Protection; Fischer F-344
1. Introduction Cells, as well as organisms, have several ways to defend themselves against injuries and stress. Acutephase responses (APR), hypothalamic±pituitary±adrenal axis responses, sympathetic autonomic nervous systems responses, oxidative stress response and integrated responses to surgical trauma are some examples of defense mechanisms [1,2]. Heat shock response (HSR), or stress response, is perhaps the oldest and most conserved form of reaction to stress. It is nearly universal in all living organisms [3]. Heating cells [4] or organisms generates the * Corresponding author. Professor T.N. Meyer, Rua Desembargador Alberto Luz, 129 37410-000 TreÃs Corac° oÄes, MG, Brazil.
expression of a class of proteins, known as heat shock proteins (HSP). Other stresses, like water immersion [5], exposure to sodium arsenite [6], dinitrophenol [4] and other metabolic poisons [7] may also elicit HSR. Some HSP act as molecular chaperones [8]: they escort other proteins during the assembling, translocation and folding process. According to their molecular masses (kD), HSP may be divided into families: HSP27, HSP47, HSP60, HSP70, HSP90 and HSP110 [3]. HSP70 is the most conserved family Ð its structure, in man, is 72% homologous to its counterpart in Drosophilae [9]. Cells and organisms previously submitted to stresses that induce HSR become protected against a second exposure to the same stress, as well as against other types of injury, provided that this happens during the period in which HSP are signi®cantly expressed. The
0305-4179/00/$20.00+0.00 # 2000 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 9 9 ) 0 0 1 3 9 - 4
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T.N. Meyer et al. / Burns 26 (2000) 233±238
HSR has important protective eects. In rabbits, HSR decreases myocardial ischemia/reperfusion injury [10]. Rat cardiomyocytes in culture are protected against further injury when preconditioned by heat shock [11]. In rats, induction of HSP70 protects the retina from light-provoked injury [12]. HSP may have cytoprotective eects on gastric mucosa [13], as well as on experimental acute ileitis [14]. Ischemic intestinal injury is reduced by previous HSP70 induction [15]. HSP70 protects rat liver against thioacetamide-induced injury [16]. HSR reduces ischemic injury in rat skeletal muscle [17]. Experimental skin ¯aps previously submitted to heat shock show increased surviving length [18] and less necrosis [19]. Also, free myocutaneous ¯aps show increased skin survival when previously conditioned by heat shock [20]. HSR reduces mortality in experimental models of septic shock and adult respiratory distress syndrome (ARDS) [21,22], and also in models of endotoxemia [23,24]. The protective potential of HSR in burns has not been investigated. In this work, we have studied the eects of previously induced HSR on mortality after severe burns in rats. Our objective was to test the hypothesis that heat shock response elicited before severe burns is protective and reduces mortality. This could have a role in future clinical applications of HSR induction. 2. Methods and materials The animal experiments carried out in the present study were performed in accordance with Ethical Committee (UNINCOR) norms and to those contained in the Guide for the care and use of laboratory animals (NIH Ð National Institutes of Health, 1996). A total of 115 adult male Fischer F-344 rats (UNINCOR, TreÃs Corac° oÄes, MG, Brasil) weighing 205±506 g were randomly assigned to four groups: C (control), n = 12; HS (heat shock), n = 15; B (burns), n = 44 and HSB (heat shock/burns), n = 44. In each group, a number of animals were used for HSP detection by Western analysis and therefore they were excluded from the mortality study. In each group, the number of animals observed for mortality was: C: n = 6; HS: n = 7; B: n = 40; HSB: n = 40 (Table 1). Groups C and HS, smaller than the other two groups, were designed to observe whether anesthesia alone or anesthesia associated with heat shock would cause deaths. All animals were placed in individual cages in the same experimental room 7 days before the procedures, with free access to standard laboratory chow and tap water. Natural light and darkness cycles were allowed. Room temperature was maintained at 288C. Follow-up observations were performed at 12-h inter-
Table 1 Groups of animals observed for mortality Group
n
Mean weight (g)
Standard deviation
C HS B HSB
6 7 40 40
368.7 369.3 291.6 292.0
17.6 46.1 39.6 47.3
vals. Mortality rates were measured at 3, 7, 15 and 25 days. After this period, the surviving animals were sacri®ced. Procedures in each group were: Group C Ð anesthesia with intraperitoneal ketamine hydrochloride (Ketalar, Parke-Davis, Guarulhos, SP, Brazil), 90 mg/kg; dorsal shaving, recovery and observation. Group HS Ð same anesthesia; heating protocol (see below), recovery and observation. Group B Ð same anesthesia; same shaving, burn protocol (see below), recovery and observation. Group HSB Ð same anesthesia; same heating protocol; 1 day (22±27 h) after heating, same anesthesia, shaving and burn protocol; recovery and observation. Animals submitted to the same procedures in all groups were sacri®ced 1, 3, 5 and 7 days after the procedures with a lethal dose of ketamine. Liver and lung samples were collected, rinsed in saline, frozen and stored in liquid nitrogen. 2.1. Heating protocol The anesthetized animal was secured to a supporting device and placed in warm water at 458C. Its rectal temperature was monitored with a digital thermometer (Digithermo, Sigma T1916) until it reached 428C. The animal was kept in the bath for an additional 5 min. 2.2. Burn protocol Burns were in¯icted on the dorsal shaved area with a 2 5.5 cm brass bar heated in boiling water and left in contact with shaved skin for 20 s. This was repeated until 26±30% of the body surface was burnt, as calculated by Meeh's formula [25]. Saline was administered by intraperitoneal injection (0.1 l/kg, or 3.6 ml/kg/% TBSA burn). 2.3. HSP detection (Western analysis) In brief, liver and lung were thawed, ®nely minced, weighed (0.22±0.23 g liver, 0.33±0.34 g lung) and homogenized in cold phosphate buered saline (PBS) containing protease inhibitors. The homogenate was
T.N. Meyer et al. / Burns 26 (2000) 233±238
centrifuged at 18,000 rpm, 4±108C for 45 min. Protein concentration in the supernatant was measured by the Biuret method (Analisa, Belo Horizonte, MG, Brazil). Samples were suspended in LaÈemmli's sample buer and submitted to heat denaturation. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS±PAGE) in a BioRad 10-well Mini-PROTEAN system (BioRad, Hercules, CA, USA), using 0.75 mm gel and 200±220 mg of total protein per lane. The positive control was a puri®ed human recombinant HSP70 (SPP-755, StressGen, Victoria, BC, Canada). After gel electrophoresis, proteins were transferred to a nitrocellulose membrane (Sigma N2764). The transfer was checked with Ponceau-S (Sigma P7170) and membranes were thoroughly washed in PBS±Tween. After nonspeci®c membrane block for 2 h at room temperature, proteins were labeled with anti-HSP70 monoclonal antibody (SPA810, StressGen) at 1:1000 concentration for 1 h at room temperature. After secondary labeling with antimouse IgG conjugated with alkaline phosphatase at 1:1000 concentration (1 h, room temperature), protein was visualized using color development with Nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma FAST BCIP/NBT, B 5655) in alkaline phosphatase buer. Blots were scanned for densitometric analysis. 2.4. Statistical analysis Data were analyzed statistically using the Winks 4.5 software (TexaSoft, evaluation version, www.share ware.com) in an IBM PC-compatible computer. Fisher's exact tests were applied to contingency tables (2 2) to compare mortality rates between groups B and HSB.
235
Fig. 1. Comparison of mortality rates in groups B (burns) and HSB (heat shock and burns).(p < 0.05 at 3 days, Rp < 0.01 at 7, 15 and 25 days).
3.3. Mortality study No animals died until day 25 in groups C and HS. Mortality in group B was 40% (16 from 40 animals) at 3 days, 52.5% (21 from 40) at 7 days, 57.5% (23 from 40) at 15 days and 62.5% (25 from 40) at 25 days. Mortality in group HSB was 17.5% (seven from 40 animals) at 3 days, 20% (eight from 40) on day 7, and 27.5% (11 from 40 animals) on day 15, with no deaths thereafter (Fig. 1). Comparison between mortality rates at 3, 7, 15 and 25 days in groups B and HSB showed statistically signi®cant dierences ( p < 0.05 until 3 days, p < 0.01 at 7, 15 and 25 days). Western analysis detected HSP70 production in lungs and liver of animals from groups HS and HSB (a representative blot is shown in Fig. 2), but not in the other groups. 4. Discussion In this work, a heating protocol using a warm water bath induced HSR in a way that was sucient to impart protection to animals submitted to burns 1 day
3. Results 3.1. Burns Burns, produced by a brass bar heated in boiling water and left in contact with shaved dorsal skin for 20 s, produced third-degree lesions, as con®rmed by histopathological studies. 3.2. Heating protocol Heated animals attained an internal temperature of 408C at a mean time of 6 min (SD=1.7), 418C at 8.5 min (SD=2.3), and 428C at 12 min (SD=3.4). Mean total time for the bath was 17 min (SD=3.4). Mean ®nal internal temperature was 438C (SD=0.3).
Fig. 2. Western blot (top) and densitometric analysis (bottom) of the expression of HSP-70 in the liver (L) and lung (Lu) of rats from groups HS (heated only) and HSB (heated and burnt after one day). HSP: heat-shock protein 70 (positive control). L: liver. Lu: lung. Western blot was negative for HSP-70 in groups C (control) and B (burnt only) (not shown). Optical density is expressed in absorbance units (AU).
236
T.N. Meyer et al. / Burns 26 (2000) 233±238
after heating. This is a simple and inexpensive method for HSR induction. In this work, no animals died due to anesthesia alone (Group C) or combined with HS (Group HS). The burn model, although more laborious than the scalding method commonly used [25], allowed for a more precise graduation of burn extent according to body surface as obtained by Meeh's formula. This process produced standard animal burns that, in the extension used, provoked a mortality rate of about 60% at 25 days. Fisher F-344 rats have been found to be more susceptible to thermal injury than other, more commonly used rat strains, as Wistar and Sprague±Dawley [26]. Previously heat-shocked animals had a mortality rate that was signi®cantly lower than that of untreated animals. This ®nding corroborates those obtained by other authors [21±24,27] in other animal models. Many deaths in groups B and HSB occurred early (up to 3 days) after the burns. The animals had received saline in adequate volume (0.1 l/kg, or 3.6 ml/kg/% TBSA (total body surface area) burn). It is possible that these deaths were due to mechanisms other than hypovolemic shock. In these animals, early burn site infection was not detected. In clinical settings, this is usually observed at a later time after thermal injury. Myocardial alterations may be an important cause of immediate mortality after burns [28±30]; ¯uid replacement does not ameliorate these alterations, which are linked to problems in calcium homeostasis and reactive oxygen metabolite (ROM) production. Burns are associated with signi®cant in¯ammatory responses [31]. An acute-phase response is elicited in burns, with increasing plasma levels of interleukin(IL)-6 and tumor necrosis factor(TNF)-a [32]. Burnt mice exhibit increased levels of TNF-a, IL-1 and IL-6 [33]. Burns activate complement and liberate several mediators which may cause local and systemic lesions; vascular injury at the burn site and in the lung may occur due to neutrophil activation [34]. Plasma levels of xanthine-oxidase may be increased [35]. Four hours after a burn, skin and lung endothelial lesions can be detected, which may be associated with ROM generation [36]. The oxidative blood red cell lesion occurring in burns may be part of an ischemia/reperfusion process in burnt areas and these erythrocytes may serve as platforms for complement activation [37]. Burns bring increased synthesis of prostaglandins, thromboxanes and leukotrienes [38]. In rat burns, neutrophils may be sequestered, after their activation, in lungs, bowel, kidneys, skin and brain, generating augmented levels of hydrogen peroxide 5 h after injury [39]. Blood endotoxin levels may show a ®ve-fold increase in extensive human burns, and may appear in the circulation after burns, before burn site bacterial colo-
nization [40]. Bacterial translocation seems to be important in severe burns, and this could be related to ROM-mediated mucosal lesions [41], as well as to mesenteric ischemia due to thromboxane-A2 [42]. Endotoxin and in¯ammatory mediators liberated by burns increase the probability of the occurrence of systemic in¯ammatory response syndrome and multiple organ dysfunction syndrome [43]. HSR may modulate in¯ammatory responses. HSP inhibit production of TNF-a and IL-1 in lipopolysaccharide(LPS)-activated human monocytes and rat macrophages [44]. Heat shock protects in vivo against several injuries associated with increased production of cytokines and/or ROM [45]. HSR increases production of superoxide-dismutase in rat lung [46], as well as catalase in rats [47]. Overexpression of HSP70 may protect cells from necrosis induced by TNF-a and TNF-b [48]. Ribeiro and co-workers [27] have shown that, in an experimental sepsis model, HSR-associated mortality reduction is associated with decreasing plasma levels of TNF-a. Klosterhalfen and associates [23] have shown that HSR reduces liberation of pro-in¯ammatory cytokines and decreases apoptosis rates. In experimental endotoxic shock, HSR decreases IL-1b levels [49]. HS-treated endothelial cells become more resistant to neutrophil-induced necrosis [50]. HSR seems to precede, to reduce or to exclude acute-phase responses (APR); the genetic programs of these two kinds of responses are distinctive, exclusive and prioritized [51]. Therefore, it is possible to speculate that, in this model of severe experimental burns, HSR reduced mortality by reducing the in¯ammatory response and its deleterious eects. Since in the present study no data were obtained to support this hypothesis, further investigation of this possibility is needed. Nevertheless, it is clear that HSR signi®cantly reduced mortality after severe burns in this model. This fact points to the existence of common disease pathways between burns and other conditions, in which HSR has also been bene®cial. Furthermore, it is reasonable to suppose that HSR exerts a protective role in several pathological conditions through the same mechanism. This may be a very basic function, such as reducing protein denaturation or refolding denatured proteins. Otherwise, in¯ammatory modulation also seems to be an acceptable hypothesis, which does not exclude the chaperone function. No published data are presently available on the use of HSR to improve outcome when employed after burn injury. Improvement has been achieved in lipopolysaccharide-stimulated rats [49]. Eects of post-burn HSR have not been investigated in this work. This may be essential to show whether HSR may become an useful therapeutic tool. We are now working on pharmacological induction of HSR performed after
T.N. Meyer et al. / Burns 26 (2000) 233±238
burns, as further research on this subject may lead to future clinical applications of this important response to stress. Acknowledgements The technical assistance of Regina de FaÂtima Lopes in animal experiments is gratefully acknowledged. Animal experiments were performed in the Department of Anatomy, UNINCOR (Vale do Rio Verde University). References [1] Meyer TN. Resposta ao trauma no idoso. In: Petroianu A, Pimenta LG, editors. Cirurgia GeriaÂtrica. Rio de Janeiro: MEDSI, 1998. p. 187±204. [2] Udelsman R, Holbrook NJ. Endocrine and molecular responses to surgical stress. Curr Probl Surg 1994;31:658±720. [3] JaÈaÈttelaÈ M, Wissing D. Emerging role of heat shock proteins in biology and medicine. Ann Med 1992;24:249±58. [4] Ritossa F. A new pung pattern induced by temperature shock and DNP in Drosophila. Experientia 1962;18:571±3. [5] Otaka M, Itoh H, Kuwabara T, et al. Induction of heat shock protein and prevention of caerulein-induced pancreatitis by water-immersion stress in rats. Int J Biochem 1994;26:805±11. [6] Ribeiro SP, Villar G, Downey GP, et al. Sodium arsenite induces heat shock protein-72 kilodalton expression in the lungs and protects rats against sepsis. Crit Care Med 1994;22:922±9. [7] Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev 1992;72:1063±81. [8] Ellis RJ. Proteins as molecular chaperones. Nature 1987;328:378±9. [9] Hunt C, Morimoto RI. Conserved features of eukaryotic hsp genes revealed by comparison with the nucleotide sequence of human hsp70. Proc Natl Acad Sci USA 1985;82:6455±9. [10] Currie RW, Tanguay RM, Kingma JG. Heat shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 1993;87:963±71. [11] Nayeem MA, Hess ML, Qian YZ, et al. Delayed preconditioning of cultured adult rat cardiac myocytes: role of 70- and 90kDa heat stress proteins. Am J Physiol 1997;273:H861±H868. [12] Barbe MF, Tytell M, Gower DJ, et al. Hyperthermia protects against light damage in the rat retina. Science (Wash DC) 1988;241:1817±20. [13] Hahm KB, Park IS, Kim YS, et al. Role of rebapimide on induction of heat-shock proteins and protection agains reactive oxygen metabolites-mediated cell damage in cultured gastric mucosal cells. Free Radic Biol Med 1997;22:711±6. [14] Stojadinovic A, Kiang J, Goldhill J, et al. Induction of heat shock response prevents tissue injury during acute in¯ammation of the rat ileum. Crit Care Med 1997;25:309±17. [15] ToÈns C, Klosterhalfen B, Klein HM, et al. Induction of heat shock protein 70 (HSP70) by zinc-bis-(DL-hydrogenaspartate) reduces ischemic small-bowel tissue damage in rats. Langenbecks Arch Chir 1997;382:43±8. [16] Fujimori S, Otaka M, Otani S, et al. Induction of a 72-kDa heat shock protein and cytoprotection against thioacetamideinduced liver injury in rats. Dig Dis Sci 1997;42:1987±94. [17] Garramone Jr RR, Winters RM, Das DK, et al. Reduction of skeletal muscle injury through stress conditioning using the heat-shock response. Plast Reconstr Surg 1994;93:1242±7.
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[18] Koenig WJ, Lohner RA, Perdrizet GA, et al. Improving acute skin-¯ap survival through stress conditioning using heat shock and recovery. Plast Reconstr Surg 1992;90:659±64. [19] RoÈsken F, RuÈcker M, Bauer I, et al. Heat shock priming reduces microvascular perfusion failure of critically perfused skin ¯aps Ð a possible mechanism of tissue protection in acute ischemia. Langenbecks Arch Chir 1997;114:527±30. [20] Wang BH, Caiying Y, Stagg CA, et al. Improved free musculocutaneous ¯ap survival with induction of heat shock protein. Plast Reconstr Surg 1998;101:776±84. [21] Villar J, Edelson JD, Post M, et al. Induction of heat stress proteins is associated with decreased mortality in an animal model of acute lung injury. Am Rev Respir Dis 1993;147:177±81. [22] Villar J, Ribeiro SP, Mullen JBM, et al. Induction of the heat shock response reduces mortality rate and organ damage in a sepsis-induced acute lung injury model. Crit Care Med 1994;22:914±21. [23] Klosterhalfen B, Hauptmann S, Oner FA, et al. Induction of heat shock protein 70 by zinc-bis-(DL-hydrogenaspartate) reduces cytokine liberation, apoptosis, and mortality rate in a rat model of LD100 endotoxemia. Shock 1997;7:254±62. [24] Klosterhalfen B, Hauptmann S, Tietze L, et al. The in¯uence of heat shock protein 70 on hemodynamic variables in a porcine model of recurrent endotoxemia. Shock 1997;7:358±63. [25] Walker HL, Mason Jr AD. A standard animal burn. J Trauma 1968;8:1049±51. [26] Rapaport FT, Bachvaro RJ, Grullon J, et al. Genetics of natural resistance to thermal injury. Ann Surg 1982;195:294±304. [27] Ribeiro SP, Villar J, Downey GP, et al. Eects of the stress response in septic rats and LPS-stimulated alveolar macrophages: evidence for TNF-a posttranslational regulation. Am J Respir Crit Care Med 1996;154:1843±50. [28] Garcia N, Horton JW, Walker P, et al. The eects of thermal injury on myocardial metabolism and nitrate excretion. FASEB J 1993;7:A322±A327. [29] Horton JW. Oxygen free radicals contribute to postburn cardiac cell membrane dysfunction. J Surg Res 1996;61:97±102. [30] Horton JW, White DJ. Diminished cardiac contractile response to burn injury in aged guinea pigs. J Trauma 1993;34:429±36. [31] Cone JB, Wallace BH, Lubansky HJ, et al. Manipulation of the in¯ammatory response to burn injury. J Trauma 1997;43:41±6. [32] Drost AC. Plasma cytokines after thermal injury and their relationship to infection. Ann Surg 1993;218:74. [33] O'Riordain MG, Collins KH, Pilz M, et al. Modulation of macrophage hyperactivity improves survival in a burn-sepsis model. Arch Surg 1992;127:152±8. [34] Mulligan MS, Till GO, Smith CW, et al. Role of leukocyte adhesion molecules in lung and dermal vascular injury after thermal trauma of skin. Am J Pathol 1994;144:1008±15. [35] Till GO, Beauchamp C, Menapace D, et al. Oxygen radical dependent lung damage following thermal injury of rat skin. J Trauma 1983;23:269±77. [36] Till GO, Hatherill JR, Tourtelotte WW, et al. Lipid peroxidation and acute lung injury after thermal trauma to skin: evidence of a role for hydroxyl radical. Am J Pathol 1985;119:376± 84. [37] Solomkin JS. Neutrophil disorders in burn injury: complement, cytokines, and organ injury. J Trauma 1990;30(Suppl 12):S80± S85. [38] Tarnow P, Cassuto J, JoÈnsson A, et al. Eects of D-myo-inositol-1,2,6-trisphosphate on eicosanoid formation in burned skin. J Surg Res 1996;62:1±4. [39] Hansbrough JF, WikstroÈm T, Braide M, et al. Neutrophil activation and tissue neutrophil sequestration in a rat model of thermal injury. J Surg Res 1996;61:17±22. [40] Winchurch RA, Thupari JN, Munster AM. Endotoxemia in
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T.N. Meyer et al. / Burns 26 (2000) 233±238 burn patients: levels of circulating endotoxins are related to burn size. Surgery 1987;102:808±12. Ma L, Ma JW, Deitch EA, et al. Genetic susceptibility to mucosal damage leads to bacterial translocation in a murine burn model. J Trauma 1989;29:1245±51. Herndon DN, Zeigler ST. Bacterial translocation after thermal injury. Crit Care Med 1993;21(Suppl 2):S50±S54. Herndon DN, Barrow RE, Rutan RL, et al. A comparison of conservative versus early excision therapies in severely burned patients. Ann Surg 1989;209:547±53. Hall TJ. Role of hsp70 in cytokine production. Experientia 1994;50:1048±53. Jacquier-Sarlin MR, Fuller K, Dinh-Xuan AT, et al. Protective eects of hsp70 in in¯ammation. Experientia 1994;50:1031±8. Hass MA, Massaro D. Regulation of the synthesis of superoxide dismutase in ratlings during oxidant and hyperthermic stresses. J Biol Chem 1988;262:776±81.
[47] Karmazyn M, Mailer K, Currie RW. Acquisition and decay of heat-shock enhanced post-ischemic ventricular recovery. Am J Physiol 1990;259:H424±H431. [48] JaÈaÈttelaÈ M. Overexpression of major heat shock protein hsp70 inhibits tumor necrosis factor-induced activation of phospholipase A2. J Immunol 1993;151:4286±94. [49] Chu EK, Ribeiro SP, Slutsky AS. Heat stress increases survival rates in lipopolysaccharide-stimulated rats. Crit Care Med 1997;25:1727±32. [50] Wang JH, Redmond HP, Watson WG, et al. Induction of heat shock protein 72 prevents neutrophil-mediated human endothelial cell necrosis. Arch Surg 1995;130:1260±5. [51] Buchman TG, Cabin DE. Molecular biology of circulatory shock III. HepG2 cells demonstrate two patterns of shockinduced gene expression which are independent, exclusive and prioritized. Surgery 1990;108:902±11.
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Heat shock response reduces mortality after severe experimental burns Tu® Neder Meyer a,*, Alcino LaÂzaro da Silva b, EÃnio Cardillo Vieira b, Antonio Carlos Vassalo Alves b a
INCIS (Health Sciences Institute), UNINCOR (Vale do Rio Verde University), TreÃs Corac° oÄes, MG, Brazil b UFMG (Federal University of Minas Gerais), Belo Horizonte, MG, Brazil Accepted 20 July 1999
Abstract The heat shock response has imparted protective eects in animal models of septic shock and endotoxemia. This study has tested the hypothesis that it could be protective in experimental burns. One hundred and ®fteen adult male Fischer rats were randomly divided into four groups. Rats in the ®rst group (n = 12) were anesthetized and shaved. In the second group (n = 15) rats were anesthetized and heated in a 458C water bath. In the third group (n = 44), rats were anesthetized, shaved and submitted to 26±30% body surface third-degree burns using a brass bar. In the fourth group (n = 44), rats were anesthetized, heated and, 1 day after, they were burnt. Mortality rates were measured at 3, 7, 15 and 25 days. Liver and lung samples were collected from all groups for heat-shock protein 70 detection. Heat-shock protein 70 was positive in heated animals. No animals died in the ®rst or second group. Heated and burnt animals showed signi®cantly decreased mortality at days 3 ( p < 0.05, Fischer's exact test) and at days 7, 15 and 25 ( p < 0.01) after burns, when compared to unheated burnt animals. In conclusion, eliciting the heat-shock response signi®cantly reduced mortality rates in this model of experimental burns. # 2000 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Heat-shock proteins; Stress proteins; Heat-shock response; Stress response; Burns; Survival rate; Protection; Fischer F-344
1. Introduction Cells, as well as organisms, have several ways to defend themselves against injuries and stress. Acutephase responses (APR), hypothalamic±pituitary±adrenal axis responses, sympathetic autonomic nervous systems responses, oxidative stress response and integrated responses to surgical trauma are some examples of defense mechanisms [1,2]. Heat shock response (HSR), or stress response, is perhaps the oldest and most conserved form of reaction to stress. It is nearly universal in all living organisms [3]. Heating cells [4] or organisms generates the * Corresponding author. Professor T.N. Meyer, Rua Desembargador Alberto Luz, 129 37410-000 TreÃs Corac° oÄes, MG, Brazil.
expression of a class of proteins, known as heat shock proteins (HSP). Other stresses, like water immersion [5], exposure to sodium arsenite [6], dinitrophenol [4] and other metabolic poisons [7] may also elicit HSR. Some HSP act as molecular chaperones [8]: they escort other proteins during the assembling, translocation and folding process. According to their molecular masses (kD), HSP may be divided into families: HSP27, HSP47, HSP60, HSP70, HSP90 and HSP110 [3]. HSP70 is the most conserved family Ð its structure, in man, is 72% homologous to its counterpart in Drosophilae [9]. Cells and organisms previously submitted to stresses that induce HSR become protected against a second exposure to the same stress, as well as against other types of injury, provided that this happens during the period in which HSP are signi®cantly expressed. The
0305-4179/00/$20.00+0.00 # 2000 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 9 9 ) 0 0 1 3 9 - 4
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T.N. Meyer et al. / Burns 26 (2000) 233±238
HSR has important protective eects. In rabbits, HSR decreases myocardial ischemia/reperfusion injury [10]. Rat cardiomyocytes in culture are protected against further injury when preconditioned by heat shock [11]. In rats, induction of HSP70 protects the retina from light-provoked injury [12]. HSP may have cytoprotective eects on gastric mucosa [13], as well as on experimental acute ileitis [14]. Ischemic intestinal injury is reduced by previous HSP70 induction [15]. HSP70 protects rat liver against thioacetamide-induced injury [16]. HSR reduces ischemic injury in rat skeletal muscle [17]. Experimental skin ¯aps previously submitted to heat shock show increased surviving length [18] and less necrosis [19]. Also, free myocutaneous ¯aps show increased skin survival when previously conditioned by heat shock [20]. HSR reduces mortality in experimental models of septic shock and adult respiratory distress syndrome (ARDS) [21,22], and also in models of endotoxemia [23,24]. The protective potential of HSR in burns has not been investigated. In this work, we have studied the eects of previously induced HSR on mortality after severe burns in rats. Our objective was to test the hypothesis that heat shock response elicited before severe burns is protective and reduces mortality. This could have a role in future clinical applications of HSR induction. 2. Methods and materials The animal experiments carried out in the present study were performed in accordance with Ethical Committee (UNINCOR) norms and to those contained in the Guide for the care and use of laboratory animals (NIH Ð National Institutes of Health, 1996). A total of 115 adult male Fischer F-344 rats (UNINCOR, TreÃs Corac° oÄes, MG, Brasil) weighing 205±506 g were randomly assigned to four groups: C (control), n = 12; HS (heat shock), n = 15; B (burns), n = 44 and HSB (heat shock/burns), n = 44. In each group, a number of animals were used for HSP detection by Western analysis and therefore they were excluded from the mortality study. In each group, the number of animals observed for mortality was: C: n = 6; HS: n = 7; B: n = 40; HSB: n = 40 (Table 1). Groups C and HS, smaller than the other two groups, were designed to observe whether anesthesia alone or anesthesia associated with heat shock would cause deaths. All animals were placed in individual cages in the same experimental room 7 days before the procedures, with free access to standard laboratory chow and tap water. Natural light and darkness cycles were allowed. Room temperature was maintained at 288C. Follow-up observations were performed at 12-h inter-
Table 1 Groups of animals observed for mortality Group
n
Mean weight (g)
Standard deviation
C HS B HSB
6 7 40 40
368.7 369.3 291.6 292.0
17.6 46.1 39.6 47.3
vals. Mortality rates were measured at 3, 7, 15 and 25 days. After this period, the surviving animals were sacri®ced. Procedures in each group were: Group C Ð anesthesia with intraperitoneal ketamine hydrochloride (Ketalar, Parke-Davis, Guarulhos, SP, Brazil), 90 mg/kg; dorsal shaving, recovery and observation. Group HS Ð same anesthesia; heating protocol (see below), recovery and observation. Group B Ð same anesthesia; same shaving, burn protocol (see below), recovery and observation. Group HSB Ð same anesthesia; same heating protocol; 1 day (22±27 h) after heating, same anesthesia, shaving and burn protocol; recovery and observation. Animals submitted to the same procedures in all groups were sacri®ced 1, 3, 5 and 7 days after the procedures with a lethal dose of ketamine. Liver and lung samples were collected, rinsed in saline, frozen and stored in liquid nitrogen. 2.1. Heating protocol The anesthetized animal was secured to a supporting device and placed in warm water at 458C. Its rectal temperature was monitored with a digital thermometer (Digithermo, Sigma T1916) until it reached 428C. The animal was kept in the bath for an additional 5 min. 2.2. Burn protocol Burns were in¯icted on the dorsal shaved area with a 2 5.5 cm brass bar heated in boiling water and left in contact with shaved skin for 20 s. This was repeated until 26±30% of the body surface was burnt, as calculated by Meeh's formula [25]. Saline was administered by intraperitoneal injection (0.1 l/kg, or 3.6 ml/kg/% TBSA burn). 2.3. HSP detection (Western analysis) In brief, liver and lung were thawed, ®nely minced, weighed (0.22±0.23 g liver, 0.33±0.34 g lung) and homogenized in cold phosphate buered saline (PBS) containing protease inhibitors. The homogenate was
T.N. Meyer et al. / Burns 26 (2000) 233±238
centrifuged at 18,000 rpm, 4±108C for 45 min. Protein concentration in the supernatant was measured by the Biuret method (Analisa, Belo Horizonte, MG, Brazil). Samples were suspended in LaÈemmli's sample buer and submitted to heat denaturation. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS±PAGE) in a BioRad 10-well Mini-PROTEAN system (BioRad, Hercules, CA, USA), using 0.75 mm gel and 200±220 mg of total protein per lane. The positive control was a puri®ed human recombinant HSP70 (SPP-755, StressGen, Victoria, BC, Canada). After gel electrophoresis, proteins were transferred to a nitrocellulose membrane (Sigma N2764). The transfer was checked with Ponceau-S (Sigma P7170) and membranes were thoroughly washed in PBS±Tween. After nonspeci®c membrane block for 2 h at room temperature, proteins were labeled with anti-HSP70 monoclonal antibody (SPA810, StressGen) at 1:1000 concentration for 1 h at room temperature. After secondary labeling with antimouse IgG conjugated with alkaline phosphatase at 1:1000 concentration (1 h, room temperature), protein was visualized using color development with Nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma FAST BCIP/NBT, B 5655) in alkaline phosphatase buer. Blots were scanned for densitometric analysis. 2.4. Statistical analysis Data were analyzed statistically using the Winks 4.5 software (TexaSoft, evaluation version, www.share ware.com) in an IBM PC-compatible computer. Fisher's exact tests were applied to contingency tables (2 2) to compare mortality rates between groups B and HSB.
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Fig. 1. Comparison of mortality rates in groups B (burns) and HSB (heat shock and burns).(p < 0.05 at 3 days, Rp < 0.01 at 7, 15 and 25 days).
3.3. Mortality study No animals died until day 25 in groups C and HS. Mortality in group B was 40% (16 from 40 animals) at 3 days, 52.5% (21 from 40) at 7 days, 57.5% (23 from 40) at 15 days and 62.5% (25 from 40) at 25 days. Mortality in group HSB was 17.5% (seven from 40 animals) at 3 days, 20% (eight from 40) on day 7, and 27.5% (11 from 40 animals) on day 15, with no deaths thereafter (Fig. 1). Comparison between mortality rates at 3, 7, 15 and 25 days in groups B and HSB showed statistically signi®cant dierences ( p < 0.05 until 3 days, p < 0.01 at 7, 15 and 25 days). Western analysis detected HSP70 production in lungs and liver of animals from groups HS and HSB (a representative blot is shown in Fig. 2), but not in the other groups. 4. Discussion In this work, a heating protocol using a warm water bath induced HSR in a way that was sucient to impart protection to animals submitted to burns 1 day
3. Results 3.1. Burns Burns, produced by a brass bar heated in boiling water and left in contact with shaved dorsal skin for 20 s, produced third-degree lesions, as con®rmed by histopathological studies. 3.2. Heating protocol Heated animals attained an internal temperature of 408C at a mean time of 6 min (SD=1.7), 418C at 8.5 min (SD=2.3), and 428C at 12 min (SD=3.4). Mean total time for the bath was 17 min (SD=3.4). Mean ®nal internal temperature was 438C (SD=0.3).
Fig. 2. Western blot (top) and densitometric analysis (bottom) of the expression of HSP-70 in the liver (L) and lung (Lu) of rats from groups HS (heated only) and HSB (heated and burnt after one day). HSP: heat-shock protein 70 (positive control). L: liver. Lu: lung. Western blot was negative for HSP-70 in groups C (control) and B (burnt only) (not shown). Optical density is expressed in absorbance units (AU).
236
T.N. Meyer et al. / Burns 26 (2000) 233±238
after heating. This is a simple and inexpensive method for HSR induction. In this work, no animals died due to anesthesia alone (Group C) or combined with HS (Group HS). The burn model, although more laborious than the scalding method commonly used [25], allowed for a more precise graduation of burn extent according to body surface as obtained by Meeh's formula. This process produced standard animal burns that, in the extension used, provoked a mortality rate of about 60% at 25 days. Fisher F-344 rats have been found to be more susceptible to thermal injury than other, more commonly used rat strains, as Wistar and Sprague±Dawley [26]. Previously heat-shocked animals had a mortality rate that was signi®cantly lower than that of untreated animals. This ®nding corroborates those obtained by other authors [21±24,27] in other animal models. Many deaths in groups B and HSB occurred early (up to 3 days) after the burns. The animals had received saline in adequate volume (0.1 l/kg, or 3.6 ml/kg/% TBSA (total body surface area) burn). It is possible that these deaths were due to mechanisms other than hypovolemic shock. In these animals, early burn site infection was not detected. In clinical settings, this is usually observed at a later time after thermal injury. Myocardial alterations may be an important cause of immediate mortality after burns [28±30]; ¯uid replacement does not ameliorate these alterations, which are linked to problems in calcium homeostasis and reactive oxygen metabolite (ROM) production. Burns are associated with signi®cant in¯ammatory responses [31]. An acute-phase response is elicited in burns, with increasing plasma levels of interleukin(IL)-6 and tumor necrosis factor(TNF)-a [32]. Burnt mice exhibit increased levels of TNF-a, IL-1 and IL-6 [33]. Burns activate complement and liberate several mediators which may cause local and systemic lesions; vascular injury at the burn site and in the lung may occur due to neutrophil activation [34]. Plasma levels of xanthine-oxidase may be increased [35]. Four hours after a burn, skin and lung endothelial lesions can be detected, which may be associated with ROM generation [36]. The oxidative blood red cell lesion occurring in burns may be part of an ischemia/reperfusion process in burnt areas and these erythrocytes may serve as platforms for complement activation [37]. Burns bring increased synthesis of prostaglandins, thromboxanes and leukotrienes [38]. In rat burns, neutrophils may be sequestered, after their activation, in lungs, bowel, kidneys, skin and brain, generating augmented levels of hydrogen peroxide 5 h after injury [39]. Blood endotoxin levels may show a ®ve-fold increase in extensive human burns, and may appear in the circulation after burns, before burn site bacterial colo-
nization [40]. Bacterial translocation seems to be important in severe burns, and this could be related to ROM-mediated mucosal lesions [41], as well as to mesenteric ischemia due to thromboxane-A2 [42]. Endotoxin and in¯ammatory mediators liberated by burns increase the probability of the occurrence of systemic in¯ammatory response syndrome and multiple organ dysfunction syndrome [43]. HSR may modulate in¯ammatory responses. HSP inhibit production of TNF-a and IL-1 in lipopolysaccharide(LPS)-activated human monocytes and rat macrophages [44]. Heat shock protects in vivo against several injuries associated with increased production of cytokines and/or ROM [45]. HSR increases production of superoxide-dismutase in rat lung [46], as well as catalase in rats [47]. Overexpression of HSP70 may protect cells from necrosis induced by TNF-a and TNF-b [48]. Ribeiro and co-workers [27] have shown that, in an experimental sepsis model, HSR-associated mortality reduction is associated with decreasing plasma levels of TNF-a. Klosterhalfen and associates [23] have shown that HSR reduces liberation of pro-in¯ammatory cytokines and decreases apoptosis rates. In experimental endotoxic shock, HSR decreases IL-1b levels [49]. HS-treated endothelial cells become more resistant to neutrophil-induced necrosis [50]. HSR seems to precede, to reduce or to exclude acute-phase responses (APR); the genetic programs of these two kinds of responses are distinctive, exclusive and prioritized [51]. Therefore, it is possible to speculate that, in this model of severe experimental burns, HSR reduced mortality by reducing the in¯ammatory response and its deleterious eects. Since in the present study no data were obtained to support this hypothesis, further investigation of this possibility is needed. Nevertheless, it is clear that HSR signi®cantly reduced mortality after severe burns in this model. This fact points to the existence of common disease pathways between burns and other conditions, in which HSR has also been bene®cial. Furthermore, it is reasonable to suppose that HSR exerts a protective role in several pathological conditions through the same mechanism. This may be a very basic function, such as reducing protein denaturation or refolding denatured proteins. Otherwise, in¯ammatory modulation also seems to be an acceptable hypothesis, which does not exclude the chaperone function. No published data are presently available on the use of HSR to improve outcome when employed after burn injury. Improvement has been achieved in lipopolysaccharide-stimulated rats [49]. Eects of post-burn HSR have not been investigated in this work. This may be essential to show whether HSR may become an useful therapeutic tool. We are now working on pharmacological induction of HSR performed after
T.N. Meyer et al. / Burns 26 (2000) 233±238
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