- Email: [email protected]
The optimal time for early burn wound excision to reduce pro-inflammatory cytokine production in a murine burn injury model
burns 36 (2010) 1059–1066
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
The optimal time for early burn wound excision to reduce pro-inflammatory cytokine production in a murine burn injury model§ Ko-Chang Chang, Hsu Ma *, Wen-Chieh Liao, Chih-Kang Lee, Chia-Yi Lin, Chen-chien Chen Division of Plastic and Reconstructive Surgery, Department of Surgery, Taipei Veteran’s General Hospital, Taiwan
article info
abstract
Article history:
Background: A potential solution to prevent post-burn deleterious inflammatory responses
Accepted 19 February 2010
is early burn wound excision. However, the most beneficial time point remains controversial. This animal study investigated the optimal time point for burn wound excision to
Keywords:
reduce pro-inflammatory cytokines production after burn.
Burn wound
Methods: Forty-eight male Sprague-Dawley rats received scald burns with third-degree
Early excision
burns of 30% body surface area, and were then divided into eight groups by day of operation
Cytokine
for excision. Group 1 (n = 6) received burn eschar excision on post-burn day (PBD) 1. Group 2
Animal study
received excision on PBD2 (n = 6) and so on, while group 8 was the control group (n = 6) that did not undergo excision. The skin defect after excision was covered with a bovine-derived collagen dressing. Interleukin-1 (IL-1), IL-6, IL-10 and tumour necrosis factor-a were serially analysed by enzyme-linked immunosorbent assay (ELISA). Results: We found that levels of all pro-inflammatory cytokines appeared to be lower after excision of full-thickness burns, but as the excision time was delayed from group 1 to group 7, the differences showed progressive decline. Conclusions: We believe that the earlier the excision is performed, the more the level of proinflammatory cytokines can be lowered, and the better the post-burn inflammatory process can be modulated. # 2010 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Major burns are associated with an acute and persistent postburn response, including changes in vascular permeability, alteration in the coagulation system, impairment of gut function, hypermetabolic response and immune depression [1]. These acute inflammatory responses are believed to be mediated by pro-inflammatory cytokines with a concomitant depression of the anti-inflammatory component. Interleukin-1
(IL-1) is an endogenous pyrogen that induces a variety of acutephase reactions [2]. Tumour necrosis factor-a (TNF-a), or cachectin, is thought to be a primary mediator of the host response to inflammation [3]. Interleukin-6 (IL-6), originally identified as a B-cell differentiation factor, is thought to be an indicator of severity in the infectious phase, while interleukin10 (IL-10) is regarded as the anti-inflammatory cytokine [4]. Thus, previous studies have suggested IL-1, IL-6, IL-10 and TNFa can reflect the severity of the morbid condition after burn [5].
§ This paper has been presented at the 14th Congress of the Internal Society of Burn Injuries (ISBI), held in Montreal Canada, on September 2008. * Corresponding author at: No. 201, 2nd Section, Shih-Pai Road, Taipei 112, Taiwan. Tel.: +886 2 2875 7542; fax: +886 2 2875 7041. E-mail addresses: [email protected] (K.-C. Chang), [email protected], [email protected] (H. Ma), [email protected] (W.-C. Liao), [email protected] (C.-K. Lee), [email protected] (C.-Y. Lin). 0305-4179/$36.00 # 2010 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2010.02.004
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The burn wound is considered to be a major source of inflammatory mediators that initiate and maintain the postburn inflammatory response, and is characterised by hypermetabolism and immunosuppression. Hypermetabolic response with increased circulating levels of catabolic hormones and development of insulin resistance will result in raised energy requirement, muscle wasting and nitrogen imbalance [6,7]. A wide variety of immunological changes following thermal injury is exhibited, including impaired neutrophil function. In a previous animal study, neutrophils were found to be less sensitive to zymosan-activated serum infusion after 30% burns compared with sham burns, and rats with greater injury have fewer neutrophils in their wounds [8]. Susceptibility to wound infection and sepsis might be increased since prompt recruitment and delivery of neutrophils to a site of bacterial invasion is of prime importance in controlling infection. Decreased lymphocyte blastogenesis and impairment of interleukin-2 production were also found in previous studies [9]. It is generally accepted that burn trauma results in the suppression of both mechanical and immunological protection against sepsis. To prevent these deleterious changes, a good solution appears to be to excise the burn wound before inflammatory response is maximised. Previous studies have suggested early burn wound excision and grafting, a surgical procedure performed to remove the burn wound eschar and cover the defect with skin graft or artificial skin substitutes [10–14]. When compared with conservative wound treatment with serial debridement and delayed grafting, early surgical intervention is associated with decreased blood loss [10], diminished wound infection [11,12] and shortened hospital stay [13,14]. Thus, early excision of full-thickness burn wound and immediate resurfacing have been considered the treatment of choice in the management of burns. However, the most beneficial time point for early excision remains controversial. Herndon et al. suggested that early excision within 48 h is optimal for paediatric patients with massive burns [15], while other studies have described early excision in the range of 24 h to 7 days after a burn [10,14,16,17]. Due to ethical concerns, a prospective controlled clinical trial to investigate the optimal time point for burn wound excision is prohibited. Thus, we designed an animal study that compares post-operative changes of IL-1, IL-6, IL-10 and TNF-a, after burn in Sprague-Dawley rats, to determine the optimal time point at which burn wound excision can reduce pro-inflammatory cytokines by modulating inflammatory and metabolic responses.
from the back by an electric shaver. The exposed skin was then scrubbed with Hibiscrub solution and 70% ethanol. The rat is then placed on its back in a mouldable metal wire cage, having their backs submerged in water at 90 8C for 20 s. This produced a full-thickness burn wound with an estimated 30% of body surface area (BSA), which was calculated by Meeh’s formula [18]. All rats were scalded and then divided into eight groups: group 1: burn eschar excision on post-burn day (PBD) 1 (n = 6); group 2: excision on PBD2 (n = 6); group 3: excision on PBD3 (n = 6); group 4: excision on PBD4 (n = 6); group 5: excision on PBD5 (n = 6); group 6: excision on PBD6 (n = 6); group 7: excision on PBD7 (n = 6); while group 8 was the control group (n = 6) that did not undergo excision following burn. All animals were resuscitated with an intra-peritoneal injection of lactated Ringer’s solution 2 mL kg 1% total body surface area (TBSA) immediately after the burn. In the excision groups (group 1 to group 7), incision was made with scalpel along the edges of burn eschar. After dissection with scissors, a plane can be easily identified between the cutaneous eschar and panniculus carnosus. The eschar was unroofed with minimal blood loss because the incision was mainly on the eschar. No apparent difference was found between different groups, in terms of blood loss, adhesion of burn wound and underlying wound base. After excision of full-thickness burn eschar, the wound was covered with Skintemp1 (BioCore Medical Technologies, Topeka, KS, USA), a biological dressing that provides a porous bovine-derived collagen membrane, tailored to fit the skin defect. Running sutures with 4/0 nylon was used to attach the dressing to the wound firmly. Rats were housed in individual cages after excision. One-millilitre blood samples were obtained from the tail prior to the burn on PBD1, 3, 5 and 7 days and on postexcision days 1, 3, 5 and 7 and then collected in endotoxin-free ethylene-diamine tetra-acetic acid containing blood specimen tubes. The samples were kept on ice and then centrifuged at 3000 rpm for 60 s within 30 min of collection. The plasma was stored at 70 8C. Cytokines were analysed by enzyme-linked immunosorbent assay (ELISA). The IL-1, IL-6, IL-10 and TNF-a kits were purchased from Bender Medsystem (Austria, Europe). Statistical analysis was performed with the Mann–Whitney U-test for inter-group comparison. Comparisons before and after the excision were made by the Wilcoxon signed-rank test. Statistical significance was defined at p < 0.05 level for all comparisons.
3. 2.
Results
Materials and methods
This animal study was approved by the Approval of Animal Use Protocol of Taipei Veterans General Hospital. Male Sprague-Dawley rats (300–350 g) were housed at 23 8C and 60% relative humidity with a 12-h light/dark cycle, with lights on at 07:00 h. Animals had free access to food (Laboratory Rodent Diet 50011, Purina Mills, Richmond, IN, USA) and water, and were used after a minimum acclimatisation period of 7 days. The rats were anaesthetised intraperitoneally with ketamine 100 mg kg 1. Hair was removed
All wounds appeared dry and clean 1 week after excision. The biological dressing was adhered firmly to the wound without signs of dehiscence or infection. No apparent difference was discerned in the resected eschars that were sent for pathological studies, which showed that necrotic levels involved full-thickness skin and partial panniculus carnosus of subcutaneous muscle (Fig. 1). When comparing post-operative cytokines levels with pre-operative levels, we found that levels of IL-1, IL-6 and TNF-a were lower after the excision. However, the difference
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Fig. 1 – Frozen sections of resected eschars showed that necrotic level involved full-thickness dermis (D) and partial panniculus carnousus (arrow).
Fig. 3 – Cytokines cascades in control group and group 1 (IL1, IL-10 and TNF-a in pg/dL; IL-6 in ng/dL). showed progressive decline from group 1 to group 7, as the excision time was delayed and only the decline of TNF-a in group 1 reached statistical significance (Fig. 2). Both IL-1 and TNF-a decreased most in group 1, that is, the group with immediate resection of the burn wound within 24 h of the burn. To look further into group 1, cytokine cascades in the control group were drawn and compared with group 1. In the control group, IL-6 and TNF-a reached a plateau at postburn day 1, while IL-1 had its peak value at post-burn day 5. In group 1, all pro-inflammatory cytokines declined after operation (Fig. 3). When comparing post-operative 5th day of group 1 with post-burn 5th day of control group, we observed that all pro-inflammatory cytokines were significantly lower (Fig. 4).
In all groups, there were no significant differences in the serum levels of IL-1, IL-6, IL-10 and TNF-a either before or 24 h after the excision. However, 5 days after the excision in group 1, a significant decrease was observed in serum levels of IL-1, IL-6 and TNF-a (Fig. 5). Levels of IL-10 were slightly higher 5 days after the excision. The same trend was also observed in group 2, but only the decline of TNF-a is statistically significant (Fig. 6). From group 3 to group 7, there were no significant differences in serum levels of all cytokines before or 5 days after the excision, except for the decrease of IL-1 in group 4 (Figs. 7–11). Cytokine levels are interrelated and we
Fig. 2 – Post-operative change of serum levels of all cytokines, compared with pre-operative levels, in percentage: levels of IL-1, and TNF-a were lower after the excision, but the difference showed progressive decline, as the excision time was delayed. Asterisk indicates p < 0.05 for comparisons among groups.
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Fig. 4 – Comparisons between group 1 post-OP 5th day and control post-burn 5th day; IL-1, IL-6 and TNF-a were lower in group 1. Asterisk indicates p < 0.05 for group 1 vs. control group.
Fig. 5 – Serum levels of all cytokines in group 1, before operation, post-operative day (POD) 1, 3, 5 and day 7: a significant decrease was observed in serum levels of IL-1, IL-6, and TNF-a 5 days after burn wound excision. Levels of IL-10 were slightly higher after excision. Asterisk indicates p < 0.05 for POD5 vs. PODI and pre-operatively.
found a weak positive correlation between IL-1 and IL-6 (r = 0.4571).
4.
Discussion
Acute inflammatory and metabolic responses are often observed after major burns, with over-production of proinflammatory cytokines and depression of anti-inflammatory cytokines that trigger a series of acute-phase reactions. Increase in pro-inflammatory cytokines and acute-phase proteins raise energy requirements, cause nitrogen imbalance
and may contribute to the development of insulin resistance and other metabolic derangement [19]. There is also evidence that IL-1, IL-6 and TNF-a stimulate release of somatostatin, which, in turn, inhibits the release of growth hormone (GH) [20–22]. When this post-burn inflammatory response is prolonged and aggravated, it results in multiple-organ failure. Various treatments have proven to be efficient in modulating acute-phase reaction and hypermetabolic responses after major burns. Wilmore et al. measured the mean metabolic rate of patients with major burns and found it significantly decreases when the room temperature is increased from 25 to 33 8C [23]. Other interventions include
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Fig. 6 – Serum levels of all cytokines in group 2, before operation, on POD1, 3, 5 and day 7: only the decline of serum levels of TNF-a on POD5 is significant statistically. Asterisk indicates p < 0.05 POD5 vs. POD1 and pre-operatively.
Fig. 7 – Serum levels of all cytokines in group 3, before operation, on POD1, 3, 5 and day 7.
Fig. 8 – Serum levels of all cytokines in group 4, before operation, on POD1, 3, 5 and day 7; IL-1 decreased significantly 5 days after burn wound excision. Asterisk indicates p < 0.05 and POD5 vs. POD1 and pre-operatively.
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Fig. 9 – Serum levels of all cytokines in group 5, before operation, on POD1, 3, 5 and day 7.
Fig. 10 – Serum levels of all cytokines in group 6, before operation, on POD1, 3, 5 and day 7.
Fig. 11 – Serum levels of all cytokines in group 7, before operation, on POD1, 3, 5 and day 7.
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nutritional modulation through early enteral supplementation [24] and hormonal modulation. In prospective randomised clinical trials, the administration of GH has been proven to increase protein synthesis and improve wound healing [25,26]. Other anabolic agents, such as IGF-1 with IGFBP-3 and oxandrolone, have been tested and have proven to be effective with fewer adverse effects [27–29]. However, none of these treatments are as effective as surgery in improving survival and outcome in major burns. Conservative wound treatment with topical agents (primarily silver sulphadiazine) by waiting for spontaneous eschar separation will cause severe metabolic derangement and multiple septic episodes. Besides, it will also prolong hospitalisation with much pain and suffering. Leaving devitalised tissue on the wound not only increases bacterial and fungal colonisation, but also induces increased bacterial and fungal invasion into subcutaneous viable tissue. This could further impair the host immune system and facilitate the induction of other routes of bacterial invasion, such as bacterial translocation [30]. By contrast, the excision of the burn eschar breaks the vicious cycle, removes the source of inflammation, reestablishes skin barrier by wound coverage and helps prevent further bacterial or fungal infection while restoring cellular immunity [31]. Despite the established benefits of early burn wound excision, the optimal time point has not been identified. Some studies have suggested 24 h to 7 days after a burn [10,14,16,17], while other studies have questioned the safety and efficacy of wound excision within 48 h of injury [32]. Potential adverse effects include increased blood loss [33], sacrifice of otherwise viable tissue or, alternatively, incomplete excision of the eschar, since the margin and depth of the burn wound are less welldemarcated and cannot be evaluated accurately [34]. Controlled prospective trials in humans are difficult because of ethical issues and the presence of many variables (e.g., age, sex, extent of injury). Thus, we designed this animal study to investigate the optimal time point at which burn wound excision reduces pro-inflammatory cytokines. In our study, the effect of early burn wound excision on reducing pro-inflammatory cytokine production was also noted. Levels of all pro-inflammatory cytokines appeared to be lower after excision of full-thickness burns, but the difference showed progressive decline from group 1 to group 7, as the excision time was delayed. Levels of IL-10, known as the anti-inflammatory cytokine, were slightly higher after the excision. Both IL-6 and TNF-a decreased most in group 1, while IL-1 decreased most in group 4. This may be due to the fact that IL-1 peaked at PBD4–5, unlike other cytokines that have peak values in the first PBDs [35]. The same trend can also be observed in the cytokine cascade of our control group. Proinflammatory cytokines peaked after burn and reached a plateau without tapering off. When comparing the post-operative 5th day of group 1 with pre-operative levels, we found that all pro-inflammatory cytokines were significantly lower, indicating a decrease in inflammatory status, which was consistent with the clinical trial by Herndon et al. in severely burned children [36]. However, there was no significant decline in the serum levels of all cytokines on the first post-operative day. This can be explained by the newly formed inflammation caused by
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surgical trauma, which neutralises the anti-inflammatory effect of eschar excision. When comparing post-operative 5th day of group 1 with post-burn 5th day of control group, we could find that all pro-inflammatory cytokines were significantly lower in the excision group. These might illustrate the benefit of prompt eschar excision after burn. Another benefit of very early burn wound excision was demonstrated by Tchervenkov et al. [37] In his experiment, the in vivo delivery of neutrophils to a bacterial skin lesion is returned to normal when the burn wound is excised 1 day following trauma. Waiting 3 days before excision improved the in vivo neutrophil influx but did not completely return it to normal. As the excision time was delayed, the beneficial effect decreased. The same tendency was also observed in our study. We found a weak positive correlation between IL-1 and IL-6. The positive correlation we observed is consistent with the fact that IL-6 can be induced by IL-1 and TNF-a [4]. However, further studies examining the precise time course after thermal injury will be necessary to confirm the interaction between these cytokines.
5.
Conclusions
In the present study, we found that immediate excision of fullthickness burn eschar on the first post-burn day can reduce pro-inflammatory cytokines significantly in rats with 30% TBSA burn injuries. Thus, we believe that the earlier the excision is performed, the more pro-inflammatory cytokines can be lowered, and the better the post-burn inflammatory process can be modulated. Further studies with a larger sample size might be required to determine the individual effects of the burn wound-healing process on the immunosuppression following burn trauma, before claiming its benefits for clinical application.
Conflict of interests statement This work was supported by the National Science Council, Taiwan (NSC95-2314-B-075-029). No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
Acknowledgement This work was supported by the National Science Council, Taiwan (NSC95-2314-B-075-029).
references
[1] Nguyen TT, Gilpin DA, Meyer NA, Herndon DN. Current treatment of severely burned patients. Ann Surg 1996;223:14–25. [2] Dinarello CA. Interleukin-1. Rev Infect Dis 1984;6:51. [3] Beutler B, Cerami A. The biology of cachetin/TNF-a primary mediator of the host response. Ann Rev Immunol 1989;7:625.
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[4] Drost AC, Burleson D, Cioffi W, Mason AD, Pruitt BA. Plasma cytokines after thermal injury and their relationship to infection. Ann Surg 1993;1:218. [5] Drost AC, Burleson D, Cioffi W, Jordan BS, Mason AD, Pruitt BA. Plasma cytokines following thermal injury and their relationship with patient mortality, burn size, and time postburn. J Trauma 1993;3:35. [6] Petersen SR, Jeevanandam M, Shahbazain LM, Holaday NJ. Reprioritization of liver protein synthesis resulting from recombinant human growth hormone supplementation in parenteral fed trauma patients: the effect of growth hormone on the acute-phase response. J Trauma 1997;42:987–96. [7] Wilmore DW, Long JM, Mason AD. Catecholamines: mediators of the hypermetabolic response to thermal burn patients. Ann Surg 1974;180:653–60. [8] Yurt RW, Pruitt Jr BA. Decreased wound neutrophils and indiscrete margination in the pathogenesis of wound infection. Surgery 1985;98:191–8. [9] Ninnemann JL. Immunologic defenses against infection: alterations following thermal injuries. J Burn Care Rehabil 1982;3:355–66. [10] Desai MH, Herndon DN, Broemeling L, Barrow RE, Nichols RJ, Rutan RL. Early burn wound excision significantly reduces blood loss. Ann Surg 1990;211:753–62. [11] Tompkins RG, Burke JF, Schoenfeld DA, Bondoc CC, Behringer GC, Ackroyd FW, et al. Prompt eschar excision: a treatment system contributing to reduced burn mortality: a statistical evaluation of burn care at the Massachusetts General Hospital (1974–1984). Ann Surg 1986;204:272–81. [12] Peck MD, Heimbach DM. Does early excision of burn wounds change the pattern of mortality? J Burn Care Rehabil 1989;10:7–10. [13] Herndon DN, Parks DN. Comparison of serial debridement and autographing and early massive excision with cadaver skin overlay in the treatment of large burns in children. J Trauma 1986;26:149–52. [14] Pietsch JB, Netscher DT, Nagaraj HS, Groff DB. Early excision of major burns in children: effect on morbidity and mortality. J Pediatr Surg 1985;20:754–7. [15] Xiao-Wu W, Herndon DN, Spies M, Sanford AP, Wolf SE. Effects of delayed wound excision and grafting in severely burned children. Arch Surg 2002;137:1049–54. [16] Irei M, Abston S, Bonds E, Rutan T, Desai MH, Herndon DN, et al. The optimal time for excision of scald burns in toddlers. J Burn Care Rehabil 1986;7:508–10. [17] Demling RH. Improved survival after massive burns. J Trauma 1983;23:179–84. [18] Walker HL, Mason Jr AD. A standard animal burn. J Trauma 1968;8:1049–51. [19] Mandrup-Poulsen T, Nerup J, Reimers JI, Pociot F, Andersen HU, Karlsen A, et al. Cytokines and the endocrine system. Eur J Endocrinol 1996;134:21–30. [20] Scarborough DE. Somatostatin regulation by cytokines. Metabolism 1990;39:108–11. [21] Jones TH, Kennedy RL. Cytokines and hypothalamic: pituitary function. Cytokine 1993;5:531–8.
[22] Scarborough DE. Cytokine modulation of pituitary hormone secretion. Ann NY Acad Sci 1990;594:169–87. [23] Wilmore DW, Long JM, Mason AD, Skreen RW, Pruitt BA. Catecholamines: mediator of the hypermetabolic response to thermal injury. Ann Surg 1974;180:653–69. [24] McDonald WS, Sharp CW, Deicht EA. Immediate enteral feeding in burn patients is safe and effective. Ann Surg 1991;213:177–83. [25] Gore DC, Honeycutt D, Jahoor F, Wolfe RR, Herndon DN. Effect of exogenous growth hormone on whole-body and isolated limb protein kinetics in burned patients. Arch Surg 1991;126:38–43. [26] Gore DC, Honeycutt D, Jahoor F, Rutan T, Wolfe RR, Herndon DN. Effect of exogenous growth hormone on glucose utilization in burn patients. J Surg Res 1991;51:518–23. [27] Herndon DN, Ramzy PI, DebRoy MA, Zheng M, Ferrando AA, Chinkes DL, et al. Muscle protein catabolism after severe burn: effects of IGF-1/IGFBP-3 treatment. Ann Surg 1999;229:713–22. [28] Jeschke MG, Barrow RE, Herndon DN. Insulinlike growth factor 1 plus insulinlike growth factor binding protein 3 attenuates the proinflammatory acute phase response in severely burned children. Ann Surg 2000;231:246–52. [29] Hart DW, Wolf SE, Ramzy PI, Chinkes DL, Beauford RB, Ferrando AA, et al. Anabolic effects of oxandrolone after severe burn. Ann Surg 2001;233:556–64. [30] Baron P, Traber LD, Traber DL, Nguyen T, Hollyoak M, Heggers JP, et al. Gut failure and translocation following burn and sepsis. J Surg Res 1994;57:197–204. [31] Cetinkale O, Ulualp KM, Ayan F, Duren M, Cizmeci O, Pusane A. Early wound excision and skin grafting restores cellular immunity after severe burn trauma. Br J Surg 1993;80:1296–8. [32] Han TH, Lee SY, Kwon JE, Kwak IS, Kim KM. The limited immunomodulatory effects of escharectomy on the kinetics of endotoxin, cytokines, and adhesion molecules in major burns. Mediat Inflamm 2004;13:241–6. [33] Herndon DN, Barrow RE, Rutan RL, Rutan TC, Desai MH, Abston S. A comparison of conservative vs early excision therapies in severely burned patients. Ann Surg 1989;209:547–53. [34] Desai MH, Rutan RL, Herndon DN. Conservative treatment of scald burns is superior to early excision. J Burn Care Rehabil 1991;12:482–4. [35] Caldwell FT, Graves DB, Wallace BH. The effect of indomethacin on the cytokine cascade and body temperature following burn injury in rats. Burns 1999;25:283–94. [36] Barret JP, Herndon DN. Modulation of inflammatory and catabolic responses in severely burned children by early burn wound excision in the first 24 hours. Arch Surg 2003;138:127–32. [37] Tchervenkov JI, Epstein MD, Silberstein EB, Alexander JW. Early burn wound excision and skin grafting postburn trauma restores in vivo neutrophil delivery to inflammatory lesions. Arch Surg 1988;123:1477–81.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
The optimal time for early burn wound excision to reduce pro-inflammatory cytokine production in a murine burn injury model§ Ko-Chang Chang, Hsu Ma *, Wen-Chieh Liao, Chih-Kang Lee, Chia-Yi Lin, Chen-chien Chen Division of Plastic and Reconstructive Surgery, Department of Surgery, Taipei Veteran’s General Hospital, Taiwan
article info
abstract
Article history:
Background: A potential solution to prevent post-burn deleterious inflammatory responses
Accepted 19 February 2010
is early burn wound excision. However, the most beneficial time point remains controversial. This animal study investigated the optimal time point for burn wound excision to
Keywords:
reduce pro-inflammatory cytokines production after burn.
Burn wound
Methods: Forty-eight male Sprague-Dawley rats received scald burns with third-degree
Early excision
burns of 30% body surface area, and were then divided into eight groups by day of operation
Cytokine
for excision. Group 1 (n = 6) received burn eschar excision on post-burn day (PBD) 1. Group 2
Animal study
received excision on PBD2 (n = 6) and so on, while group 8 was the control group (n = 6) that did not undergo excision. The skin defect after excision was covered with a bovine-derived collagen dressing. Interleukin-1 (IL-1), IL-6, IL-10 and tumour necrosis factor-a were serially analysed by enzyme-linked immunosorbent assay (ELISA). Results: We found that levels of all pro-inflammatory cytokines appeared to be lower after excision of full-thickness burns, but as the excision time was delayed from group 1 to group 7, the differences showed progressive decline. Conclusions: We believe that the earlier the excision is performed, the more the level of proinflammatory cytokines can be lowered, and the better the post-burn inflammatory process can be modulated. # 2010 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Major burns are associated with an acute and persistent postburn response, including changes in vascular permeability, alteration in the coagulation system, impairment of gut function, hypermetabolic response and immune depression [1]. These acute inflammatory responses are believed to be mediated by pro-inflammatory cytokines with a concomitant depression of the anti-inflammatory component. Interleukin-1
(IL-1) is an endogenous pyrogen that induces a variety of acutephase reactions [2]. Tumour necrosis factor-a (TNF-a), or cachectin, is thought to be a primary mediator of the host response to inflammation [3]. Interleukin-6 (IL-6), originally identified as a B-cell differentiation factor, is thought to be an indicator of severity in the infectious phase, while interleukin10 (IL-10) is regarded as the anti-inflammatory cytokine [4]. Thus, previous studies have suggested IL-1, IL-6, IL-10 and TNFa can reflect the severity of the morbid condition after burn [5].
§ This paper has been presented at the 14th Congress of the Internal Society of Burn Injuries (ISBI), held in Montreal Canada, on September 2008. * Corresponding author at: No. 201, 2nd Section, Shih-Pai Road, Taipei 112, Taiwan. Tel.: +886 2 2875 7542; fax: +886 2 2875 7041. E-mail addresses: [email protected] (K.-C. Chang), [email protected], [email protected] (H. Ma), [email protected] (W.-C. Liao), [email protected] (C.-K. Lee), [email protected] (C.-Y. Lin). 0305-4179/$36.00 # 2010 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2010.02.004
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The burn wound is considered to be a major source of inflammatory mediators that initiate and maintain the postburn inflammatory response, and is characterised by hypermetabolism and immunosuppression. Hypermetabolic response with increased circulating levels of catabolic hormones and development of insulin resistance will result in raised energy requirement, muscle wasting and nitrogen imbalance [6,7]. A wide variety of immunological changes following thermal injury is exhibited, including impaired neutrophil function. In a previous animal study, neutrophils were found to be less sensitive to zymosan-activated serum infusion after 30% burns compared with sham burns, and rats with greater injury have fewer neutrophils in their wounds [8]. Susceptibility to wound infection and sepsis might be increased since prompt recruitment and delivery of neutrophils to a site of bacterial invasion is of prime importance in controlling infection. Decreased lymphocyte blastogenesis and impairment of interleukin-2 production were also found in previous studies [9]. It is generally accepted that burn trauma results in the suppression of both mechanical and immunological protection against sepsis. To prevent these deleterious changes, a good solution appears to be to excise the burn wound before inflammatory response is maximised. Previous studies have suggested early burn wound excision and grafting, a surgical procedure performed to remove the burn wound eschar and cover the defect with skin graft or artificial skin substitutes [10–14]. When compared with conservative wound treatment with serial debridement and delayed grafting, early surgical intervention is associated with decreased blood loss [10], diminished wound infection [11,12] and shortened hospital stay [13,14]. Thus, early excision of full-thickness burn wound and immediate resurfacing have been considered the treatment of choice in the management of burns. However, the most beneficial time point for early excision remains controversial. Herndon et al. suggested that early excision within 48 h is optimal for paediatric patients with massive burns [15], while other studies have described early excision in the range of 24 h to 7 days after a burn [10,14,16,17]. Due to ethical concerns, a prospective controlled clinical trial to investigate the optimal time point for burn wound excision is prohibited. Thus, we designed an animal study that compares post-operative changes of IL-1, IL-6, IL-10 and TNF-a, after burn in Sprague-Dawley rats, to determine the optimal time point at which burn wound excision can reduce pro-inflammatory cytokines by modulating inflammatory and metabolic responses.
from the back by an electric shaver. The exposed skin was then scrubbed with Hibiscrub solution and 70% ethanol. The rat is then placed on its back in a mouldable metal wire cage, having their backs submerged in water at 90 8C for 20 s. This produced a full-thickness burn wound with an estimated 30% of body surface area (BSA), which was calculated by Meeh’s formula [18]. All rats were scalded and then divided into eight groups: group 1: burn eschar excision on post-burn day (PBD) 1 (n = 6); group 2: excision on PBD2 (n = 6); group 3: excision on PBD3 (n = 6); group 4: excision on PBD4 (n = 6); group 5: excision on PBD5 (n = 6); group 6: excision on PBD6 (n = 6); group 7: excision on PBD7 (n = 6); while group 8 was the control group (n = 6) that did not undergo excision following burn. All animals were resuscitated with an intra-peritoneal injection of lactated Ringer’s solution 2 mL kg 1% total body surface area (TBSA) immediately after the burn. In the excision groups (group 1 to group 7), incision was made with scalpel along the edges of burn eschar. After dissection with scissors, a plane can be easily identified between the cutaneous eschar and panniculus carnosus. The eschar was unroofed with minimal blood loss because the incision was mainly on the eschar. No apparent difference was found between different groups, in terms of blood loss, adhesion of burn wound and underlying wound base. After excision of full-thickness burn eschar, the wound was covered with Skintemp1 (BioCore Medical Technologies, Topeka, KS, USA), a biological dressing that provides a porous bovine-derived collagen membrane, tailored to fit the skin defect. Running sutures with 4/0 nylon was used to attach the dressing to the wound firmly. Rats were housed in individual cages after excision. One-millilitre blood samples were obtained from the tail prior to the burn on PBD1, 3, 5 and 7 days and on postexcision days 1, 3, 5 and 7 and then collected in endotoxin-free ethylene-diamine tetra-acetic acid containing blood specimen tubes. The samples were kept on ice and then centrifuged at 3000 rpm for 60 s within 30 min of collection. The plasma was stored at 70 8C. Cytokines were analysed by enzyme-linked immunosorbent assay (ELISA). The IL-1, IL-6, IL-10 and TNF-a kits were purchased from Bender Medsystem (Austria, Europe). Statistical analysis was performed with the Mann–Whitney U-test for inter-group comparison. Comparisons before and after the excision were made by the Wilcoxon signed-rank test. Statistical significance was defined at p < 0.05 level for all comparisons.
3. 2.
Results
Materials and methods
This animal study was approved by the Approval of Animal Use Protocol of Taipei Veterans General Hospital. Male Sprague-Dawley rats (300–350 g) were housed at 23 8C and 60% relative humidity with a 12-h light/dark cycle, with lights on at 07:00 h. Animals had free access to food (Laboratory Rodent Diet 50011, Purina Mills, Richmond, IN, USA) and water, and were used after a minimum acclimatisation period of 7 days. The rats were anaesthetised intraperitoneally with ketamine 100 mg kg 1. Hair was removed
All wounds appeared dry and clean 1 week after excision. The biological dressing was adhered firmly to the wound without signs of dehiscence or infection. No apparent difference was discerned in the resected eschars that were sent for pathological studies, which showed that necrotic levels involved full-thickness skin and partial panniculus carnosus of subcutaneous muscle (Fig. 1). When comparing post-operative cytokines levels with pre-operative levels, we found that levels of IL-1, IL-6 and TNF-a were lower after the excision. However, the difference
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Fig. 1 – Frozen sections of resected eschars showed that necrotic level involved full-thickness dermis (D) and partial panniculus carnousus (arrow).
Fig. 3 – Cytokines cascades in control group and group 1 (IL1, IL-10 and TNF-a in pg/dL; IL-6 in ng/dL). showed progressive decline from group 1 to group 7, as the excision time was delayed and only the decline of TNF-a in group 1 reached statistical significance (Fig. 2). Both IL-1 and TNF-a decreased most in group 1, that is, the group with immediate resection of the burn wound within 24 h of the burn. To look further into group 1, cytokine cascades in the control group were drawn and compared with group 1. In the control group, IL-6 and TNF-a reached a plateau at postburn day 1, while IL-1 had its peak value at post-burn day 5. In group 1, all pro-inflammatory cytokines declined after operation (Fig. 3). When comparing post-operative 5th day of group 1 with post-burn 5th day of control group, we observed that all pro-inflammatory cytokines were significantly lower (Fig. 4).
In all groups, there were no significant differences in the serum levels of IL-1, IL-6, IL-10 and TNF-a either before or 24 h after the excision. However, 5 days after the excision in group 1, a significant decrease was observed in serum levels of IL-1, IL-6 and TNF-a (Fig. 5). Levels of IL-10 were slightly higher 5 days after the excision. The same trend was also observed in group 2, but only the decline of TNF-a is statistically significant (Fig. 6). From group 3 to group 7, there were no significant differences in serum levels of all cytokines before or 5 days after the excision, except for the decrease of IL-1 in group 4 (Figs. 7–11). Cytokine levels are interrelated and we
Fig. 2 – Post-operative change of serum levels of all cytokines, compared with pre-operative levels, in percentage: levels of IL-1, and TNF-a were lower after the excision, but the difference showed progressive decline, as the excision time was delayed. Asterisk indicates p < 0.05 for comparisons among groups.
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Fig. 4 – Comparisons between group 1 post-OP 5th day and control post-burn 5th day; IL-1, IL-6 and TNF-a were lower in group 1. Asterisk indicates p < 0.05 for group 1 vs. control group.
Fig. 5 – Serum levels of all cytokines in group 1, before operation, post-operative day (POD) 1, 3, 5 and day 7: a significant decrease was observed in serum levels of IL-1, IL-6, and TNF-a 5 days after burn wound excision. Levels of IL-10 were slightly higher after excision. Asterisk indicates p < 0.05 for POD5 vs. PODI and pre-operatively.
found a weak positive correlation between IL-1 and IL-6 (r = 0.4571).
4.
Discussion
Acute inflammatory and metabolic responses are often observed after major burns, with over-production of proinflammatory cytokines and depression of anti-inflammatory cytokines that trigger a series of acute-phase reactions. Increase in pro-inflammatory cytokines and acute-phase proteins raise energy requirements, cause nitrogen imbalance
and may contribute to the development of insulin resistance and other metabolic derangement [19]. There is also evidence that IL-1, IL-6 and TNF-a stimulate release of somatostatin, which, in turn, inhibits the release of growth hormone (GH) [20–22]. When this post-burn inflammatory response is prolonged and aggravated, it results in multiple-organ failure. Various treatments have proven to be efficient in modulating acute-phase reaction and hypermetabolic responses after major burns. Wilmore et al. measured the mean metabolic rate of patients with major burns and found it significantly decreases when the room temperature is increased from 25 to 33 8C [23]. Other interventions include
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Fig. 6 – Serum levels of all cytokines in group 2, before operation, on POD1, 3, 5 and day 7: only the decline of serum levels of TNF-a on POD5 is significant statistically. Asterisk indicates p < 0.05 POD5 vs. POD1 and pre-operatively.
Fig. 7 – Serum levels of all cytokines in group 3, before operation, on POD1, 3, 5 and day 7.
Fig. 8 – Serum levels of all cytokines in group 4, before operation, on POD1, 3, 5 and day 7; IL-1 decreased significantly 5 days after burn wound excision. Asterisk indicates p < 0.05 and POD5 vs. POD1 and pre-operatively.
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Fig. 9 – Serum levels of all cytokines in group 5, before operation, on POD1, 3, 5 and day 7.
Fig. 10 – Serum levels of all cytokines in group 6, before operation, on POD1, 3, 5 and day 7.
Fig. 11 – Serum levels of all cytokines in group 7, before operation, on POD1, 3, 5 and day 7.
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nutritional modulation through early enteral supplementation [24] and hormonal modulation. In prospective randomised clinical trials, the administration of GH has been proven to increase protein synthesis and improve wound healing [25,26]. Other anabolic agents, such as IGF-1 with IGFBP-3 and oxandrolone, have been tested and have proven to be effective with fewer adverse effects [27–29]. However, none of these treatments are as effective as surgery in improving survival and outcome in major burns. Conservative wound treatment with topical agents (primarily silver sulphadiazine) by waiting for spontaneous eschar separation will cause severe metabolic derangement and multiple septic episodes. Besides, it will also prolong hospitalisation with much pain and suffering. Leaving devitalised tissue on the wound not only increases bacterial and fungal colonisation, but also induces increased bacterial and fungal invasion into subcutaneous viable tissue. This could further impair the host immune system and facilitate the induction of other routes of bacterial invasion, such as bacterial translocation [30]. By contrast, the excision of the burn eschar breaks the vicious cycle, removes the source of inflammation, reestablishes skin barrier by wound coverage and helps prevent further bacterial or fungal infection while restoring cellular immunity [31]. Despite the established benefits of early burn wound excision, the optimal time point has not been identified. Some studies have suggested 24 h to 7 days after a burn [10,14,16,17], while other studies have questioned the safety and efficacy of wound excision within 48 h of injury [32]. Potential adverse effects include increased blood loss [33], sacrifice of otherwise viable tissue or, alternatively, incomplete excision of the eschar, since the margin and depth of the burn wound are less welldemarcated and cannot be evaluated accurately [34]. Controlled prospective trials in humans are difficult because of ethical issues and the presence of many variables (e.g., age, sex, extent of injury). Thus, we designed this animal study to investigate the optimal time point at which burn wound excision reduces pro-inflammatory cytokines. In our study, the effect of early burn wound excision on reducing pro-inflammatory cytokine production was also noted. Levels of all pro-inflammatory cytokines appeared to be lower after excision of full-thickness burns, but the difference showed progressive decline from group 1 to group 7, as the excision time was delayed. Levels of IL-10, known as the anti-inflammatory cytokine, were slightly higher after the excision. Both IL-6 and TNF-a decreased most in group 1, while IL-1 decreased most in group 4. This may be due to the fact that IL-1 peaked at PBD4–5, unlike other cytokines that have peak values in the first PBDs [35]. The same trend can also be observed in the cytokine cascade of our control group. Proinflammatory cytokines peaked after burn and reached a plateau without tapering off. When comparing the post-operative 5th day of group 1 with pre-operative levels, we found that all pro-inflammatory cytokines were significantly lower, indicating a decrease in inflammatory status, which was consistent with the clinical trial by Herndon et al. in severely burned children [36]. However, there was no significant decline in the serum levels of all cytokines on the first post-operative day. This can be explained by the newly formed inflammation caused by
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surgical trauma, which neutralises the anti-inflammatory effect of eschar excision. When comparing post-operative 5th day of group 1 with post-burn 5th day of control group, we could find that all pro-inflammatory cytokines were significantly lower in the excision group. These might illustrate the benefit of prompt eschar excision after burn. Another benefit of very early burn wound excision was demonstrated by Tchervenkov et al. [37] In his experiment, the in vivo delivery of neutrophils to a bacterial skin lesion is returned to normal when the burn wound is excised 1 day following trauma. Waiting 3 days before excision improved the in vivo neutrophil influx but did not completely return it to normal. As the excision time was delayed, the beneficial effect decreased. The same tendency was also observed in our study. We found a weak positive correlation between IL-1 and IL-6. The positive correlation we observed is consistent with the fact that IL-6 can be induced by IL-1 and TNF-a [4]. However, further studies examining the precise time course after thermal injury will be necessary to confirm the interaction between these cytokines.
5.
Conclusions
In the present study, we found that immediate excision of fullthickness burn eschar on the first post-burn day can reduce pro-inflammatory cytokines significantly in rats with 30% TBSA burn injuries. Thus, we believe that the earlier the excision is performed, the more pro-inflammatory cytokines can be lowered, and the better the post-burn inflammatory process can be modulated. Further studies with a larger sample size might be required to determine the individual effects of the burn wound-healing process on the immunosuppression following burn trauma, before claiming its benefits for clinical application.
Conflict of interests statement This work was supported by the National Science Council, Taiwan (NSC95-2314-B-075-029). No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
Acknowledgement This work was supported by the National Science Council, Taiwan (NSC95-2314-B-075-029).
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