- Email: [email protected]
Saving the zone of stasis in burns with activated protein C: An experimental study in rats
burns 36 (2010) 397–402
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
Saving the zone of stasis in burns with activated protein C: An experimental study in rats Mustafa Nisanci a,*, Muhitdin Eski a, Ismail Sahin a, Seyfettin Ilgan b, Selcuk Isik a a
Department of Plastic and Reconstructive Surgery and Burn Center, Gulhane Military Medical Academy and Medical School, 06018 Etlik, Ankara, Turkey b Department of Nuclear Medicine, Gulhane Military Medical Academy and Medical School, 06018 Etlik, Ankara, Turkey
article info
abstract
Article history:
Salvage of the zone of stasis is a major subject of focus in burn research. Use of various
Accepted 24 June 2009
antithrombotic, anti-inflammatory and antioxidant drugs have been studied experimentally, with reports of favourable results; however, none became popular in clinical practice.
Keywords:
Activated protein C (APC) is a well-known physiologic anticoagulant. Recent studies
Burn injury
revealed that APC contributes not only to systemic anticoagulant activities but also to anti-
Saving the zone of stasis
inflammatory activities by inhibiting leucocyte activation associated with TNF production.
Activated protein C
The likely favourable effects of APC on salvage of the zone of stasis were investigated on a well-described experimental rat burn model representing the zone of stasis according to the ‘burn comb model’. Twenty Sprague-Dawley rats were used and randomly separated into experimental and control groups. Two hours after inducing injury, 100 mg kg
1
APC (Sigma,
Boehringer Ingelheim, Germany) was administered to the experimental group through the caudal vein while 0.9% saline was injected through the same route in the control group. Laser Doppler flowmetry measurements and autoradiography were used for evaluation of perfusion and viability in the zone of stasis. At day 3, the differences between the results obtained from the treatment and the control groups were found to be statistically significant ( p < 0.05). Our experimental study revealed that APC improved tissue perfusion and decreased the area of skin necrosis in the zone of stasis in rats. The dual effect of APC, each of which has been shown to be favourable in saving the zone of stasis, may make this agent effective with a single effect in treatment of burn injury. # 2009 Elsevier Ltd and ISBI. All rights reserved.
Jackson suggested a burn model, representing an initial pattern of thermal injury that could be divided into several zones [1]. Based on the severity of destruction and blood-flow alterations, three distinct zones of tissue injury can be distinguished in his model. Centrally, the zone of coagulation is characterised by coagulation necrosis with thrombosed blood vessels, representing the irreversible tissue damage by direct effect of thermal energy. The intermediate zone, encircling the central zone is the zone of stasis, yet vital but
with stagnant blood supply, leading to an ischaemic insult. The outermost layer is the zone of hyperaemia with increased blood flow, representing an inflammatory response to tissue injury. Soon after burn this pattern of tissue damage appears and persists for at least 24–48 h or longer in more severe burns [2]. Investigations on the pathobiology of the zone of stasis revealed that irreversible tissue necrosis ensues with progression of hypoxia and ischaemia in 1–48 h, resulting in total loss of this intermediate zone [3]. The reversible nature of tissue
* Corresponding author at: GATA Plastik, Rekonstru¨ktif ve Estetik Cerrahi AD., 06018 Etlik, Ankara, Turkey. Tel.: +90 312 304 54 09; fax: +90 312 304 54 04. E-mail address: [email protected] (M. Nisanci). 0305-4179/$36.00 # 2009 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2009.06.208
398
burns 36 (2010) 397–402
damage in this zone at the very early stage of the burns suggested that this zone could be salvaged when tissue necrosis associated with progressive ischaemic insult was prevented. Hence, for many years, burn research has focused on saving this zone. Both hypercoagulability and systemic activation of white blood cells were reported as underlying reasons of progressive tissue injury in this zone [4]. Relying on the experimental studies, treatment modalities, such as increasing tissue tolerance to ischaemia, enhancement of perfusion and inhibition of inflammatory response, have been proposed to enable salvage of this zone. The use of antithrombotic, anticoagulant, anti-inflammatory and antioxidant medications have all been studied, with reports of favourable results [4–10]. Although all have been demonstrated to be effective experimentally, none has become popular in clinical use [4]. Activated protein C (APC) is an important physiological anticoagulant that is generated from protein C by the action of thrombomodulin–thrombin complex on endothelial cells [11]. Recently, a growing body of evidence revealed that APC not only has systemic anticoagulant activity but also produces an anti-inflammatory effect by inhibiting leucocyte activation associated with a decrease in TNF production [11–15]. This implies that APC has a dual activity, including systemic anticoagulant and anti-inflammatory activities [11–15]. Both anticoagulant and anti-inflammatory agents have been studied on experimental models individually and each has been shown to have beneficial effects in salvage of the zone of stasis [4–9]. It may be inferred that these two distinct groups of drugs must show synergistic activity when they are combined. Accordingly, because of its dual activity, APC may offer the benefits of both anticoagulant and anti-inflammatory agents in a single drug for salvage of the zone of stasis. Conclusively, it seemed reasonable to anticipate that use of APC could prevent progression of tissue injury in this zone with its dual activity and the current study was undertaken to investigate the possible favourable effects of the APC on experimental model of the zone of stasis.
1.
Material and methods
1.1.
Experimental protocol and burn model
Twenty male Sprague-Dawley rats, weighing 300–350 g, were provided by the Experimental Research Center at the Gu¨lhane Military Medical Academy (GMMA). The animals were caged individually at room temperature with a 12 h light/dark cycle and had free access to water and standard laboratory food for rats. All animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the Ethics Council. The study and the experimental protocol were approved by the Ethics Council and Animal Research Committee of GMMA. The experimental model that could meet the needs of our study was to provide a burn in which the zones described by Jacobson could be distinguished. We used the ‘comb model’, which was deemed to be the most appropriate experimental model to produce an injury with predictable zones. This model was first described by Regas and Erhlich [10] and the technical
details of their method were well presented in their original article. Since then, their model has been well accepted and used in various studies involving the zone of stasis [6–9].
1.1.1.
Procedure
General anaesthesia was induced with intramuscular ketamine 10% (90 mg kg 1) and xylasine 2% (10 mg kg 1). The entire backs of the rats were shaved and the procedure to create a ‘comb burn’ model was carried out as described by Regas and Erhlich. A specially manufactured brass block template, containing four rows (1 cm 2 cm) and three interspaces (0.5 cm 1 cm) was immersed into boiling water until thermal equilibrium was achieved. The heated brass block was then placed on the back of the rat, 0.5 cm lateral to the midline and held for 20 s without any pressure. The same procedure was performed symmetrically, 15 min later, on the other side of the rat’s back (Fig. 1). Twenty rats were randomly separated into experimental and control groups. Two hours after induction of burn, 100 mg kg 1 APC (Sigma, Boehringer Ingelheim, Germany) was administered to the experimental group through the caudal (tail) vein while 0.9% saline was injected through the same route in the control group.
1.1.2.
Laser Doppler flowmetry
Full-thickness burns were confirmed by measuring the flow in the burned rows of the back skin using Laser Doppler flowmeter (Laserflo BPM2, Vasamedics, St Paul, MN, USA) and a skin probe (Vasamedics, cat. No: P440) with an interference depth of 1.2 mm. The blood flows in the interspaces and unburned areas (caudally at least 2 cm distant from the burned areas) were measured after induction of burn injury just before the treatment with saline or APC. Measurements were repeated at day 3 after burn before injecting a radioactive agent (Fig. 2). The blood flow values obtained from three different parts, burned rows (coagulation zone), interspaces (zone of stasis) and unburned areas, were recorded separately for each rat, and the means calculated were considered as the average blood flow value for each specific area.
1.2. Evaluation of viable area with nuclear imaging and autoradiography Autoradiography was used to depict perfusion patterns in the burned and unburned skin of the rats in both the groups. Following general anaesthesia, the rats were injected with 3 mCi of technetium-99m methoxyisobutylisonitrile (Tc-99m sestamibi) through the tail veins at third day. Thirty minutes after the injection, the rats were sacrificed by decapitation. The entire back skin, including the panniculus carnosus muscle layer, was incised and dissected (Fig. 3). The specimens were then laid on supportive film layers to prevent distortion during production of images. Images were analysed with Electronic Autoradiography System (Instantimager Electronic Autoradiography, Packard Instrument Co., Meriden, CT, USA). Total necrotic and viable area were calculated in both the groups by drawing the region of interest (ROI) over the hypoactive (necrotic) and hyperactive (viable) regions of interspaces and vertical spaces of burn rows. The total necrotic and viable areas of interspaces and vertical space
burns 36 (2010) 397–402
Fig. 1 – The comb burn model immediately after burn.
399
Fig. 3 – The burn wound was prepared for autoradiography.
area between the two groups of burn rows were counted on transferring the images to a sheet with 1-mm2 grids, and the results were expressed as percentage of necrotic and viable areas.
1.3.
Statistical methods
Average blood-flow measurements and average percentages of surviving interspaces were expressed in mean standard deviation. Mann–Whitney U test was used to make comparisons within the groups. SPSS, version 15.0 computer program, was used for analysis. Statistical significance was presumed at p < 0.05.
Fig. 4 – Comparison of the mean skin blood flow in experimental and control group. (*p < 0.05).
2.
Results
2.1.
Laser Doppler flowmetry
Normal skin blood flow was measured as 18.40 1.50 ml LD/ min/100 g tissue whereas blood flow in the burned area was found to be 0.68 0.15 ml LD/min/100 g tissue. Two hours after burn, the mean blood-flow measurement in the zone of stasis was 9.73 0.70 ml LD/min/100 g in both the groups. However, at post-burn day 3, blood-flow measurement in the zone of stasis was 9.61 0.60 ml LD/min/100 g in APC-treated group, whereas 2.12 0.58 ml LD/min/100 g in the control group, as depicted in Fig. 4. The differences between the treatment and control groups at post-burn day 3 were found to be statistically significant ( p < 0.05).
2.2. Fig. 2 – Skin blood flow was measured Laser Doppler flowmetry.
Autoradiography
The vital and necrotic areas were quantified for evaluation using autoradiography (Figs. 5 and 6). Using the sheets
400
burns 36 (2010) 397–402
Fig. 7 – Mean surviving percentage of total evaluated area in control and experimental groups. (*p < 0.05).
Fig. 5 – Autoradiography of the dorsal skin of a rat in the control group; vital and necrotic areas in intervening space are barely seen.
Fig. 6 – Autoradiography of the dorsal skin of a rat in the treatment group; note that intervening spaces are preserved.
with 1-mm2 grids, the viable areas were measured in both the groups. The mean percentages of viable area of interspaces and vertical space area were 83.00 3.63 and 29.06 2.36 in the treatment and control groups, respectively. Fig. 7 shows a comparison of the percentage of both the groups. The differences between the treatment and control groups were statistically significant ( p < 00.5).
3.
Discussion
Tissue damage in the zone of stasis is thought to be reversible as long as progression of ischaemia to necrosis could be prevented. It is proposed that this zone could be salvaged with proper interventions during the first 72 h following injury. However, subsequently, necrosis is inevitable, leading to further increase in the severity of burn injury. Hence, salvage of this zone is presumed to prevent worsening of the burn. The comb model, first described and presented by Regas and Ehrlich [10] is well established and widely used in the investigation of the zone of stasis, hence we used this experimental model to simulate a burn injury with a predictable zone of stasis [6–9]. Angiography, laser Doppler flowmetry, laser Doppler perfusion scanner, histological examination and autoradiography have all been used to demonstrate the tissue viability in the previous studies referring to the zone of stasis [6–9]. We used both laser Doppler flowmetry and autoradiography images to obtain the quantitative data that may enable us to discriminate viable areas from non-viable skin. Autoradiography has previously been shown to be able to detect the exact borders between necrotic and surviving areas [7]. This simple technique was proposed to be the method of choice, especially when spatial resolution is more important than absolute sensitivity [16]. Moreover, quantitative results obtained by digital autoradiography enabled us to compare ischaemic and surviving areas with objective measurements. Any effort to develop a new treatment modality to salvage the zone of stasis requires full knowledge of the underlying pathophysiological mechanisms leading to tissue necrosis in this zone. It was reported that progressive ischaemia due to hypoperfusion inevitably results in irreversible tissue necrosis in the zone of stasis unless prevented [17]. Moreover, decreased diameters or total blockage of vessel lumens in microcirculation were shown to be responsible for diminished perfusion and ischaemia in this zone of burn injury. Thrombosis due to the hypercoagulability that appears soon
burns 36 (2010) 397–402
after burn injury was believed to cause blockage of vessel lumens in the burn wound. The studies relying on this hypothesis revealed that recombinant tissue-type plasminogen activator (r-tPA) was able to decrease the extent of necrosis in the zone of stasis, and the anticoagulant agent, rNAPc2, could save this zone by improving the perfusion of burn wound [7,17]. In addition, neutrophil recruitment and adherence to the endothelium following burn injury decreases the lumen diameter [3]. In our previous intra-vital microcirculatory study, we have shown that burn injury causes systemic activation of leucocytes and accumulation of white cells in microcirculation (increased rolling and sticking leucocytes) [18]. This also causes stasis of microcirculation, which was one of the possible explanations of skin necrosis of the zone of stasis. Prevention of leucocyte and endothelial activation using different drugs improved the circulation [19– 21]. It appears that anticoagulant and anti-inflammatory agents may have synergistic therapeutic effects on the salvage of the zone of stasis. In this study, we have shown that APC was able to improve the perfusion in the zone of stasis and decrease the area of skin necrosis, because of its antithrombotic and anti-inflammatory properties. APC is an important physiological anticoagulant derived from protein C by the action of the thrombomodulin–thrombin complex on endothelial cells [22]. APC exhibits antithrombotic properties through inhibition of activated factors V and VIII, and profibrinolytic properties through inhibition of plasminogen activator inhibitor 1. APC, an endogenous vitamin K-dependent serine protease with multiple biological activities, is also an important modulator of the host systemic response to severe infection [23]. Recently, a growing body of evidences revealed that APC not only has systemic anticoagulant activity but also produces an anti-inflammatory effect by inhibiting leucocyte activation associated with a decrease in TNF production [11– 15]. Evidence indicated that APC may reduce tissue damage after reperfusion of an ischaemic tissue owing to an antiinflammatory activity or by a mechanism independent of its anticoagulant activity [11–13]. Although APC does not inhibit the activation of neutrophils directly, it inhibits TNF production by lipopolysaccharide (LPS)-stimulated monocytes [15]. Furthermore, APC was shown to inhibit the increase in plasma levels of TNF in rats given LPS and ischaemia/reperfusion (I/R)induced increases of renal TNF levels, suggesting that APC may inhibit TNF production and thereby inhibit neutrophil activation [11,15]. TNF, known as alarm cytokine, can initiate a systemic inflammatory response independently by inducing systemic leucocyte and endothelial cell activation. Accordingly, inhibition of TNF production may prevent the subsequent neutrophil activation in burn injury. In one of our intravital study, we showed that treatment with cerium nitrate (CN) bathing decreased the number of rolling, sticking and transmigrating leucocytes by an unknown mechanism, and it was speculated that this was probably due to a decreased level of TNF production. This suggestion was supported by our following study showing that CN treatment decreased the level of TNF production after burn injury [24]. Recombinant human activated protein C was recently approved by the Food and Drug Administration for treatment of patients with severe sepsis; the approval was based on the
401
19.4% reduction in the relative risk of death (an absolute risk reduction of 6.1%) found in the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis study [25,26]. It was reported that the incidence of thrombotic events was not increased by treatment with drotrecogin alfa activated (aPC), and the anti-inflammatory effect was not associated with an increased incidence of new infections [27]. It was reported that APC was safe and well tolerated and demonstrated a dose-dependent reduction in D-dimer and interleukin 6 levels relative to placebo in sepsis treatment in human studies [27,28]. A dose of 24 mg kg 1 h 1 for 96 h was recommended for use in clinical studies [28]. However, in animal studies, it was reported that the dose of 100 mg kg 1 APC showed both anti-inflammatory and anticoagulant effect on rats [11]. Therefore, 100 mg kg 1 was the preferred injection dose in this study. Potential complication of the drug is incidence of thrombotic events and increased incidence of new infections due to the anti-inflammatory effect. However, we did not observe this potential complication in any of the rats in the experimental group. In conclusion, APC has dual effects; it may offer the benefits of both anticoagulant and anti-inflammatory agents in a single drug. Its dual effect, each of which has been shown to be favourable in saving the zone of stasis, may make activated protein C more effective than any other agent with a single effect in treatment of burn injury. Our experimental study showed that APC has beneficial effects and can at least improve the perfusion of the zone of stasis when it is used early in burn injury. Possible mechanisms of its beneficial effect and comparison with other agents are amenable to further investigations.
Acknowledgments This study was supported by the Experimental Research Center of the Gulhane Military Medical Academy. We would like to thank Vets. Tayfun Ide and Elvin Akdag for their assistance with performing this study. Disclosure: The authors will not receive benefit of any kind either directly or indirectly and have no commercial in any of the research presented here.
references
[1] Jackson DM. The diagnosis of the depth of burning. Br J Surg 1953;40:588–96. [2] Dziewulski P. Burn wound healing: James Ellsworth Laing memorial Essay for 1991. Burns 1992;18(6):466–78. [3] Boykin JV, Eriksson E, Pittman RN. In vivo microcirculation of scald burn and the progression of postburn dermal ischaemia. Plast Reconstr Surg 1980;66:191–8. [4] Arturson G. Pathophysiology of the burn wound and pharmacological treatment: the Rudi Hermans Lecture. Burns 1996;22:255–74. [5] Kufman T, Neuman RA, Weinberg A. Is postburn dermal ischemia enhanced by oxygen free radicals? Burns 1989;15:291–4. [6] Isik S S, Kopal C, Karacalıoglu O, Selmanpakoglu N. The effect of the delay phenomenon on the zone of stasis in
402
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
[15]
[16] [17]
[18]
burns 36 (2010) 397–402
burns: experimental study in rabbits. Ann Burns Fire Disasters 2000;13:101–6. Isik S, Sahin U, Ilgan S, Guler M, Gunalp B, Selmanpakoglu N. Saving the zone of stasis in burn with recombinant tissue-type plasminogen activator (r-tPA): an experimental study in rats. Burns 1998;24:217–23. Choi M, Ehrlich HP. U75412E: a lazaroid, prevents progressive burn ischemia in a rat burn model. Am J Pathol 1993;142(2):519–28. Zor F, Ozturk S, Deveci M, Karacalioglu O, Sengezer M. Saving the zone of stasis: is glutathione effective? Burns 2005;31:972–96. Regas FC, Ehrlich HP. Elucidating the vascular response to burns with a new rat model. J Trauma 1992;32:557–63. Mizutani A, Okajima K, Uchiba M, Noguchi T. Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation. Blood 2000;95(12):3781–7. Taylor Jr FB, Chang A, Esmon CT, D‘Angelo A, ViganoD‘Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987;79(3):918–25. Taylor Jr FB, Chang AC, Peer GT, Mather T, Blick K, Catlett R, et al. DEGR-factor Xa blocks disseminated intravascular coagulation initiated by Escherichia coli without preventing shock or organ damage. Blood 1991;78:364–8. Murakami K, Okajima K, Uchiba M, Johno M, Nakagaki T, Okabe H, et al. Activated protein C attenuates endotoxininduced pulmonary vascular injury by inhibiting activated leukocytes in rats. Blood 1996;87(2):642–7. Murakami K, Okajima K, Uchiba M, Johno M, Nakagaki T, Okabe H, et al. Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production. Am J Physiol 1997;272(2Pt1):L197–202. Boyd GA. In: Boyd GA, editor. Autoradiography in Biology and Medicine. New York: Academic; 1955. Mahajan AL, Tenorio X, Pepper MS, Baetens D, Montandon D, Schlaudraff K, et al. Progressive tissue injury in burns is reduced by rNAPc2. Burns 2006;32:957–63. Eski M, Deveci M, Celikoz B, Nisanci M, Turegun M. Treatment with cerium nitrate bathing modulate systemic
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26] [27]
[28]
leukocyte activation following burn injury: an experimental study in rat cremaster muscle flap. Burns 2001;27:739–46. Chappell VL, LaGrone L, Mileski WJ. Inhibition of leukocytemediated tissue destruction by synthetic fibronectin peptide (Trp-9-Tyr). J Burn Care Rehabil 1999;20(6):505–10. Bucky LP, Vedder NB, Hong HZ, Ehrlich HP, Winn RK, Harlan JM, et al. Reduction of burn injury by inhibiting CD18-mediated leukocyte adherence in rabbits. Plast Reconstr Surg 1994;93(7):1473–80. Mileski WJ, Burkhart D, Hunt JL, Kagan RJ, Saffle JR, Herndon DN, et al. Clinical effect of inhibiting leukocyte adhesion with monoclonal antibody to intercellular adhesion molecule-1 (enlimomab) in the treatment of partial-thickness burn injury. J Trauma 2003;54(5):950–8. Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 1979;571:333–9. Griffin JH, Kojima K, Banka CL, Curtiss LK, Ferna´ndez JA. High-density lipoprotein enhancement of anticoagulant activities of plasma protein S and activated protein C. J Clin Invest 1999;103:219–27. Deveci M, Eski M, Sengezer M, Kısa U. Effect of cerium nitrate bathing and prompt burn wound excision on IL-6 and TNF-Alfa levels in burned rat. Burns 2000;26:41–5. Green C, Dinnes J, Takeda A, Shepherd J, Hartwell D, Cave C, et al. Clinical effectiveness and cost-effectiveness of drotrecogin alfa (activated) (Xigris1) for the treatment of severe sepsis in adults: a systematic review and economic evaluation. Health Technol Assess 2005;9(11). Russell JA. Management of sepsis. N Engl J Med 2006;355(October (16)):1699–713. Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344(March (10)):699–709. Bernard GR, Ely EW, Wright TJ, Fraiz J, Stasek Jr JE, Russell JA, et al. Safety and dose relationship of recombinant human activated protein C for coagulopathy in severe sepsis. Crit Care Med 2001;29(11):2051–9.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
Saving the zone of stasis in burns with activated protein C: An experimental study in rats Mustafa Nisanci a,*, Muhitdin Eski a, Ismail Sahin a, Seyfettin Ilgan b, Selcuk Isik a a
Department of Plastic and Reconstructive Surgery and Burn Center, Gulhane Military Medical Academy and Medical School, 06018 Etlik, Ankara, Turkey b Department of Nuclear Medicine, Gulhane Military Medical Academy and Medical School, 06018 Etlik, Ankara, Turkey
article info
abstract
Article history:
Salvage of the zone of stasis is a major subject of focus in burn research. Use of various
Accepted 24 June 2009
antithrombotic, anti-inflammatory and antioxidant drugs have been studied experimentally, with reports of favourable results; however, none became popular in clinical practice.
Keywords:
Activated protein C (APC) is a well-known physiologic anticoagulant. Recent studies
Burn injury
revealed that APC contributes not only to systemic anticoagulant activities but also to anti-
Saving the zone of stasis
inflammatory activities by inhibiting leucocyte activation associated with TNF production.
Activated protein C
The likely favourable effects of APC on salvage of the zone of stasis were investigated on a well-described experimental rat burn model representing the zone of stasis according to the ‘burn comb model’. Twenty Sprague-Dawley rats were used and randomly separated into experimental and control groups. Two hours after inducing injury, 100 mg kg
1
APC (Sigma,
Boehringer Ingelheim, Germany) was administered to the experimental group through the caudal vein while 0.9% saline was injected through the same route in the control group. Laser Doppler flowmetry measurements and autoradiography were used for evaluation of perfusion and viability in the zone of stasis. At day 3, the differences between the results obtained from the treatment and the control groups were found to be statistically significant ( p < 0.05). Our experimental study revealed that APC improved tissue perfusion and decreased the area of skin necrosis in the zone of stasis in rats. The dual effect of APC, each of which has been shown to be favourable in saving the zone of stasis, may make this agent effective with a single effect in treatment of burn injury. # 2009 Elsevier Ltd and ISBI. All rights reserved.
Jackson suggested a burn model, representing an initial pattern of thermal injury that could be divided into several zones [1]. Based on the severity of destruction and blood-flow alterations, three distinct zones of tissue injury can be distinguished in his model. Centrally, the zone of coagulation is characterised by coagulation necrosis with thrombosed blood vessels, representing the irreversible tissue damage by direct effect of thermal energy. The intermediate zone, encircling the central zone is the zone of stasis, yet vital but
with stagnant blood supply, leading to an ischaemic insult. The outermost layer is the zone of hyperaemia with increased blood flow, representing an inflammatory response to tissue injury. Soon after burn this pattern of tissue damage appears and persists for at least 24–48 h or longer in more severe burns [2]. Investigations on the pathobiology of the zone of stasis revealed that irreversible tissue necrosis ensues with progression of hypoxia and ischaemia in 1–48 h, resulting in total loss of this intermediate zone [3]. The reversible nature of tissue
* Corresponding author at: GATA Plastik, Rekonstru¨ktif ve Estetik Cerrahi AD., 06018 Etlik, Ankara, Turkey. Tel.: +90 312 304 54 09; fax: +90 312 304 54 04. E-mail address: [email protected] (M. Nisanci). 0305-4179/$36.00 # 2009 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2009.06.208
398
burns 36 (2010) 397–402
damage in this zone at the very early stage of the burns suggested that this zone could be salvaged when tissue necrosis associated with progressive ischaemic insult was prevented. Hence, for many years, burn research has focused on saving this zone. Both hypercoagulability and systemic activation of white blood cells were reported as underlying reasons of progressive tissue injury in this zone [4]. Relying on the experimental studies, treatment modalities, such as increasing tissue tolerance to ischaemia, enhancement of perfusion and inhibition of inflammatory response, have been proposed to enable salvage of this zone. The use of antithrombotic, anticoagulant, anti-inflammatory and antioxidant medications have all been studied, with reports of favourable results [4–10]. Although all have been demonstrated to be effective experimentally, none has become popular in clinical use [4]. Activated protein C (APC) is an important physiological anticoagulant that is generated from protein C by the action of thrombomodulin–thrombin complex on endothelial cells [11]. Recently, a growing body of evidence revealed that APC not only has systemic anticoagulant activity but also produces an anti-inflammatory effect by inhibiting leucocyte activation associated with a decrease in TNF production [11–15]. This implies that APC has a dual activity, including systemic anticoagulant and anti-inflammatory activities [11–15]. Both anticoagulant and anti-inflammatory agents have been studied on experimental models individually and each has been shown to have beneficial effects in salvage of the zone of stasis [4–9]. It may be inferred that these two distinct groups of drugs must show synergistic activity when they are combined. Accordingly, because of its dual activity, APC may offer the benefits of both anticoagulant and anti-inflammatory agents in a single drug for salvage of the zone of stasis. Conclusively, it seemed reasonable to anticipate that use of APC could prevent progression of tissue injury in this zone with its dual activity and the current study was undertaken to investigate the possible favourable effects of the APC on experimental model of the zone of stasis.
1.
Material and methods
1.1.
Experimental protocol and burn model
Twenty male Sprague-Dawley rats, weighing 300–350 g, were provided by the Experimental Research Center at the Gu¨lhane Military Medical Academy (GMMA). The animals were caged individually at room temperature with a 12 h light/dark cycle and had free access to water and standard laboratory food for rats. All animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the Ethics Council. The study and the experimental protocol were approved by the Ethics Council and Animal Research Committee of GMMA. The experimental model that could meet the needs of our study was to provide a burn in which the zones described by Jacobson could be distinguished. We used the ‘comb model’, which was deemed to be the most appropriate experimental model to produce an injury with predictable zones. This model was first described by Regas and Erhlich [10] and the technical
details of their method were well presented in their original article. Since then, their model has been well accepted and used in various studies involving the zone of stasis [6–9].
1.1.1.
Procedure
General anaesthesia was induced with intramuscular ketamine 10% (90 mg kg 1) and xylasine 2% (10 mg kg 1). The entire backs of the rats were shaved and the procedure to create a ‘comb burn’ model was carried out as described by Regas and Erhlich. A specially manufactured brass block template, containing four rows (1 cm 2 cm) and three interspaces (0.5 cm 1 cm) was immersed into boiling water until thermal equilibrium was achieved. The heated brass block was then placed on the back of the rat, 0.5 cm lateral to the midline and held for 20 s without any pressure. The same procedure was performed symmetrically, 15 min later, on the other side of the rat’s back (Fig. 1). Twenty rats were randomly separated into experimental and control groups. Two hours after induction of burn, 100 mg kg 1 APC (Sigma, Boehringer Ingelheim, Germany) was administered to the experimental group through the caudal (tail) vein while 0.9% saline was injected through the same route in the control group.
1.1.2.
Laser Doppler flowmetry
Full-thickness burns were confirmed by measuring the flow in the burned rows of the back skin using Laser Doppler flowmeter (Laserflo BPM2, Vasamedics, St Paul, MN, USA) and a skin probe (Vasamedics, cat. No: P440) with an interference depth of 1.2 mm. The blood flows in the interspaces and unburned areas (caudally at least 2 cm distant from the burned areas) were measured after induction of burn injury just before the treatment with saline or APC. Measurements were repeated at day 3 after burn before injecting a radioactive agent (Fig. 2). The blood flow values obtained from three different parts, burned rows (coagulation zone), interspaces (zone of stasis) and unburned areas, were recorded separately for each rat, and the means calculated were considered as the average blood flow value for each specific area.
1.2. Evaluation of viable area with nuclear imaging and autoradiography Autoradiography was used to depict perfusion patterns in the burned and unburned skin of the rats in both the groups. Following general anaesthesia, the rats were injected with 3 mCi of technetium-99m methoxyisobutylisonitrile (Tc-99m sestamibi) through the tail veins at third day. Thirty minutes after the injection, the rats were sacrificed by decapitation. The entire back skin, including the panniculus carnosus muscle layer, was incised and dissected (Fig. 3). The specimens were then laid on supportive film layers to prevent distortion during production of images. Images were analysed with Electronic Autoradiography System (Instantimager Electronic Autoradiography, Packard Instrument Co., Meriden, CT, USA). Total necrotic and viable area were calculated in both the groups by drawing the region of interest (ROI) over the hypoactive (necrotic) and hyperactive (viable) regions of interspaces and vertical spaces of burn rows. The total necrotic and viable areas of interspaces and vertical space
burns 36 (2010) 397–402
Fig. 1 – The comb burn model immediately after burn.
399
Fig. 3 – The burn wound was prepared for autoradiography.
area between the two groups of burn rows were counted on transferring the images to a sheet with 1-mm2 grids, and the results were expressed as percentage of necrotic and viable areas.
1.3.
Statistical methods
Average blood-flow measurements and average percentages of surviving interspaces were expressed in mean standard deviation. Mann–Whitney U test was used to make comparisons within the groups. SPSS, version 15.0 computer program, was used for analysis. Statistical significance was presumed at p < 0.05.
Fig. 4 – Comparison of the mean skin blood flow in experimental and control group. (*p < 0.05).
2.
Results
2.1.
Laser Doppler flowmetry
Normal skin blood flow was measured as 18.40 1.50 ml LD/ min/100 g tissue whereas blood flow in the burned area was found to be 0.68 0.15 ml LD/min/100 g tissue. Two hours after burn, the mean blood-flow measurement in the zone of stasis was 9.73 0.70 ml LD/min/100 g in both the groups. However, at post-burn day 3, blood-flow measurement in the zone of stasis was 9.61 0.60 ml LD/min/100 g in APC-treated group, whereas 2.12 0.58 ml LD/min/100 g in the control group, as depicted in Fig. 4. The differences between the treatment and control groups at post-burn day 3 were found to be statistically significant ( p < 0.05).
2.2. Fig. 2 – Skin blood flow was measured Laser Doppler flowmetry.
Autoradiography
The vital and necrotic areas were quantified for evaluation using autoradiography (Figs. 5 and 6). Using the sheets
400
burns 36 (2010) 397–402
Fig. 7 – Mean surviving percentage of total evaluated area in control and experimental groups. (*p < 0.05).
Fig. 5 – Autoradiography of the dorsal skin of a rat in the control group; vital and necrotic areas in intervening space are barely seen.
Fig. 6 – Autoradiography of the dorsal skin of a rat in the treatment group; note that intervening spaces are preserved.
with 1-mm2 grids, the viable areas were measured in both the groups. The mean percentages of viable area of interspaces and vertical space area were 83.00 3.63 and 29.06 2.36 in the treatment and control groups, respectively. Fig. 7 shows a comparison of the percentage of both the groups. The differences between the treatment and control groups were statistically significant ( p < 00.5).
3.
Discussion
Tissue damage in the zone of stasis is thought to be reversible as long as progression of ischaemia to necrosis could be prevented. It is proposed that this zone could be salvaged with proper interventions during the first 72 h following injury. However, subsequently, necrosis is inevitable, leading to further increase in the severity of burn injury. Hence, salvage of this zone is presumed to prevent worsening of the burn. The comb model, first described and presented by Regas and Ehrlich [10] is well established and widely used in the investigation of the zone of stasis, hence we used this experimental model to simulate a burn injury with a predictable zone of stasis [6–9]. Angiography, laser Doppler flowmetry, laser Doppler perfusion scanner, histological examination and autoradiography have all been used to demonstrate the tissue viability in the previous studies referring to the zone of stasis [6–9]. We used both laser Doppler flowmetry and autoradiography images to obtain the quantitative data that may enable us to discriminate viable areas from non-viable skin. Autoradiography has previously been shown to be able to detect the exact borders between necrotic and surviving areas [7]. This simple technique was proposed to be the method of choice, especially when spatial resolution is more important than absolute sensitivity [16]. Moreover, quantitative results obtained by digital autoradiography enabled us to compare ischaemic and surviving areas with objective measurements. Any effort to develop a new treatment modality to salvage the zone of stasis requires full knowledge of the underlying pathophysiological mechanisms leading to tissue necrosis in this zone. It was reported that progressive ischaemia due to hypoperfusion inevitably results in irreversible tissue necrosis in the zone of stasis unless prevented [17]. Moreover, decreased diameters or total blockage of vessel lumens in microcirculation were shown to be responsible for diminished perfusion and ischaemia in this zone of burn injury. Thrombosis due to the hypercoagulability that appears soon
burns 36 (2010) 397–402
after burn injury was believed to cause blockage of vessel lumens in the burn wound. The studies relying on this hypothesis revealed that recombinant tissue-type plasminogen activator (r-tPA) was able to decrease the extent of necrosis in the zone of stasis, and the anticoagulant agent, rNAPc2, could save this zone by improving the perfusion of burn wound [7,17]. In addition, neutrophil recruitment and adherence to the endothelium following burn injury decreases the lumen diameter [3]. In our previous intra-vital microcirculatory study, we have shown that burn injury causes systemic activation of leucocytes and accumulation of white cells in microcirculation (increased rolling and sticking leucocytes) [18]. This also causes stasis of microcirculation, which was one of the possible explanations of skin necrosis of the zone of stasis. Prevention of leucocyte and endothelial activation using different drugs improved the circulation [19– 21]. It appears that anticoagulant and anti-inflammatory agents may have synergistic therapeutic effects on the salvage of the zone of stasis. In this study, we have shown that APC was able to improve the perfusion in the zone of stasis and decrease the area of skin necrosis, because of its antithrombotic and anti-inflammatory properties. APC is an important physiological anticoagulant derived from protein C by the action of the thrombomodulin–thrombin complex on endothelial cells [22]. APC exhibits antithrombotic properties through inhibition of activated factors V and VIII, and profibrinolytic properties through inhibition of plasminogen activator inhibitor 1. APC, an endogenous vitamin K-dependent serine protease with multiple biological activities, is also an important modulator of the host systemic response to severe infection [23]. Recently, a growing body of evidences revealed that APC not only has systemic anticoagulant activity but also produces an anti-inflammatory effect by inhibiting leucocyte activation associated with a decrease in TNF production [11– 15]. Evidence indicated that APC may reduce tissue damage after reperfusion of an ischaemic tissue owing to an antiinflammatory activity or by a mechanism independent of its anticoagulant activity [11–13]. Although APC does not inhibit the activation of neutrophils directly, it inhibits TNF production by lipopolysaccharide (LPS)-stimulated monocytes [15]. Furthermore, APC was shown to inhibit the increase in plasma levels of TNF in rats given LPS and ischaemia/reperfusion (I/R)induced increases of renal TNF levels, suggesting that APC may inhibit TNF production and thereby inhibit neutrophil activation [11,15]. TNF, known as alarm cytokine, can initiate a systemic inflammatory response independently by inducing systemic leucocyte and endothelial cell activation. Accordingly, inhibition of TNF production may prevent the subsequent neutrophil activation in burn injury. In one of our intravital study, we showed that treatment with cerium nitrate (CN) bathing decreased the number of rolling, sticking and transmigrating leucocytes by an unknown mechanism, and it was speculated that this was probably due to a decreased level of TNF production. This suggestion was supported by our following study showing that CN treatment decreased the level of TNF production after burn injury [24]. Recombinant human activated protein C was recently approved by the Food and Drug Administration for treatment of patients with severe sepsis; the approval was based on the
401
19.4% reduction in the relative risk of death (an absolute risk reduction of 6.1%) found in the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis study [25,26]. It was reported that the incidence of thrombotic events was not increased by treatment with drotrecogin alfa activated (aPC), and the anti-inflammatory effect was not associated with an increased incidence of new infections [27]. It was reported that APC was safe and well tolerated and demonstrated a dose-dependent reduction in D-dimer and interleukin 6 levels relative to placebo in sepsis treatment in human studies [27,28]. A dose of 24 mg kg 1 h 1 for 96 h was recommended for use in clinical studies [28]. However, in animal studies, it was reported that the dose of 100 mg kg 1 APC showed both anti-inflammatory and anticoagulant effect on rats [11]. Therefore, 100 mg kg 1 was the preferred injection dose in this study. Potential complication of the drug is incidence of thrombotic events and increased incidence of new infections due to the anti-inflammatory effect. However, we did not observe this potential complication in any of the rats in the experimental group. In conclusion, APC has dual effects; it may offer the benefits of both anticoagulant and anti-inflammatory agents in a single drug. Its dual effect, each of which has been shown to be favourable in saving the zone of stasis, may make activated protein C more effective than any other agent with a single effect in treatment of burn injury. Our experimental study showed that APC has beneficial effects and can at least improve the perfusion of the zone of stasis when it is used early in burn injury. Possible mechanisms of its beneficial effect and comparison with other agents are amenable to further investigations.
Acknowledgments This study was supported by the Experimental Research Center of the Gulhane Military Medical Academy. We would like to thank Vets. Tayfun Ide and Elvin Akdag for their assistance with performing this study. Disclosure: The authors will not receive benefit of any kind either directly or indirectly and have no commercial in any of the research presented here.
references
[1] Jackson DM. The diagnosis of the depth of burning. Br J Surg 1953;40:588–96. [2] Dziewulski P. Burn wound healing: James Ellsworth Laing memorial Essay for 1991. Burns 1992;18(6):466–78. [3] Boykin JV, Eriksson E, Pittman RN. In vivo microcirculation of scald burn and the progression of postburn dermal ischaemia. Plast Reconstr Surg 1980;66:191–8. [4] Arturson G. Pathophysiology of the burn wound and pharmacological treatment: the Rudi Hermans Lecture. Burns 1996;22:255–74. [5] Kufman T, Neuman RA, Weinberg A. Is postburn dermal ischemia enhanced by oxygen free radicals? Burns 1989;15:291–4. [6] Isik S S, Kopal C, Karacalıoglu O, Selmanpakoglu N. The effect of the delay phenomenon on the zone of stasis in
402
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
[15]
[16] [17]
[18]
burns 36 (2010) 397–402
burns: experimental study in rabbits. Ann Burns Fire Disasters 2000;13:101–6. Isik S, Sahin U, Ilgan S, Guler M, Gunalp B, Selmanpakoglu N. Saving the zone of stasis in burn with recombinant tissue-type plasminogen activator (r-tPA): an experimental study in rats. Burns 1998;24:217–23. Choi M, Ehrlich HP. U75412E: a lazaroid, prevents progressive burn ischemia in a rat burn model. Am J Pathol 1993;142(2):519–28. Zor F, Ozturk S, Deveci M, Karacalioglu O, Sengezer M. Saving the zone of stasis: is glutathione effective? Burns 2005;31:972–96. Regas FC, Ehrlich HP. Elucidating the vascular response to burns with a new rat model. J Trauma 1992;32:557–63. Mizutani A, Okajima K, Uchiba M, Noguchi T. Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation. Blood 2000;95(12):3781–7. Taylor Jr FB, Chang A, Esmon CT, D‘Angelo A, ViganoD‘Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987;79(3):918–25. Taylor Jr FB, Chang AC, Peer GT, Mather T, Blick K, Catlett R, et al. DEGR-factor Xa blocks disseminated intravascular coagulation initiated by Escherichia coli without preventing shock or organ damage. Blood 1991;78:364–8. Murakami K, Okajima K, Uchiba M, Johno M, Nakagaki T, Okabe H, et al. Activated protein C attenuates endotoxininduced pulmonary vascular injury by inhibiting activated leukocytes in rats. Blood 1996;87(2):642–7. Murakami K, Okajima K, Uchiba M, Johno M, Nakagaki T, Okabe H, et al. Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production. Am J Physiol 1997;272(2Pt1):L197–202. Boyd GA. In: Boyd GA, editor. Autoradiography in Biology and Medicine. New York: Academic; 1955. Mahajan AL, Tenorio X, Pepper MS, Baetens D, Montandon D, Schlaudraff K, et al. Progressive tissue injury in burns is reduced by rNAPc2. Burns 2006;32:957–63. Eski M, Deveci M, Celikoz B, Nisanci M, Turegun M. Treatment with cerium nitrate bathing modulate systemic
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26] [27]
[28]
leukocyte activation following burn injury: an experimental study in rat cremaster muscle flap. Burns 2001;27:739–46. Chappell VL, LaGrone L, Mileski WJ. Inhibition of leukocytemediated tissue destruction by synthetic fibronectin peptide (Trp-9-Tyr). J Burn Care Rehabil 1999;20(6):505–10. Bucky LP, Vedder NB, Hong HZ, Ehrlich HP, Winn RK, Harlan JM, et al. Reduction of burn injury by inhibiting CD18-mediated leukocyte adherence in rabbits. Plast Reconstr Surg 1994;93(7):1473–80. Mileski WJ, Burkhart D, Hunt JL, Kagan RJ, Saffle JR, Herndon DN, et al. Clinical effect of inhibiting leukocyte adhesion with monoclonal antibody to intercellular adhesion molecule-1 (enlimomab) in the treatment of partial-thickness burn injury. J Trauma 2003;54(5):950–8. Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 1979;571:333–9. Griffin JH, Kojima K, Banka CL, Curtiss LK, Ferna´ndez JA. High-density lipoprotein enhancement of anticoagulant activities of plasma protein S and activated protein C. J Clin Invest 1999;103:219–27. Deveci M, Eski M, Sengezer M, Kısa U. Effect of cerium nitrate bathing and prompt burn wound excision on IL-6 and TNF-Alfa levels in burned rat. Burns 2000;26:41–5. Green C, Dinnes J, Takeda A, Shepherd J, Hartwell D, Cave C, et al. Clinical effectiveness and cost-effectiveness of drotrecogin alfa (activated) (Xigris1) for the treatment of severe sepsis in adults: a systematic review and economic evaluation. Health Technol Assess 2005;9(11). Russell JA. Management of sepsis. N Engl J Med 2006;355(October (16)):1699–713. Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344(March (10)):699–709. Bernard GR, Ely EW, Wright TJ, Fraiz J, Stasek Jr JE, Russell JA, et al. Safety and dose relationship of recombinant human activated protein C for coagulopathy in severe sepsis. Crit Care Med 2001;29(11):2051–9.