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A new model for the standardization of experimental burn wounds
burns 41 (2015) 542–547
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/burns
A new model for the standardization of experimental burn wounds Neil G. Venter a,*, Andre´a Monte-Alto-Costa b, Ruy G. Marques a a
Department of Surgery, Rio de Janeiro State University, Boulevard Vinte e Oito de Setembro, 77, Vila Isabel, Rio de Janeiro, RJ 20551-030, Brazil b Department of Histology and Embryology, Rio de Janeiro State University, Av. Marechal Rondon, 381/HLA, Sa˜o Francisco Xavier, Rio de Janeiro, RJ 20950-003, Brazil
article info
abstract
Article history:
Background: Burns are common and recurrent events treated by physicians on a daily basis
Accepted 7 August 2014
at most emergency rooms around the world. There is a constant need to understand the physiopathology of burns, so as to minimize their devastating results. The objective of the
Keywords:
present report is to describe a burn apparatus in association with an innovative method of
Experimental model
animal fixation, as to produce burns of varying sizes and depths.
Burn
Methods: Rats were subjected to burns of 60 8C, 70 8C, and 80 8C for 10 s and after 3 days half of the rats in each group were killed and the resulting lesions were analyzed using histological techniques. In the other half of the rats the wound was measured weakly until complete re-epithelialization. Results: All burns were easily visible and the histological feature for the 60 8C burn was a superficial second-degree burn (28% of the dermis), for 70 8C we observed a deep seconddegree burn (72% of the dermis), and in the 80 8C group, a third degree-burn was present (100% of the dermis). Conclusions: This is a safe, reliable, easy to construct and use model that has the ability to produce a regular and uniform reproducible burn due to precise temperature control associated with standardized animal positioning. # 2014 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Burns are among the most common injuries in modern life [1]. In the United States alone, every year 450,000 patients receive medical treatment related to burns, with over 40,000 hospital admissions and 3500 deaths due to burns [2].
Initial studies concerning burns were focused on improving the overall survival rate. These works provided a better understanding of the physiopathology of burns and led to enhanced resuscitation techniques that drastically reduced the number of deaths of burn victims during the early phases of injury. In a second phase, the focus of investigation shifted toward minimizing morbidity and improving the quality of life.
* Corresponding author at: Av. Ayrton Senna, 170/2008 Barra da Tijuca, Rio de Janeiro, RJ 22793-000, Brazil. Tel.: +55 21 997797500; fax: +55 21 37964417. E-mail addresses: [email protected] (N.G. Venter), [email protected] (A. Monte-Alto-Costa), [email protected] (R.G. Marques). Abbreviations: C, Celsius; V, Volts. http://dx.doi.org/10.1016/j.burns.2014.08.002 0305-4179/# 2014 Elsevier Ltd and ISBI. All rights reserved.
burns 41 (2015) 542–547
Nowadays, one of the primary approaches concerns scar quality, especially when the burn does not affect a large body surface [3]. Therefore, in experimental models, the burn areas must be small enough not to have a systemic repercussion, but yet large enough to permit adequate observation and sampling. An experimental model is essential to test therapies before clinical use. In order to evaluate the effectiveness of burn wound treatment, however, it is extremely important to be able to create predictable and uniform burns, where most of the confounding variables can be eliminated. Burn depth, defined by three different elements – temperature, time of exposure, and contact pressure – is the primary determinant of prognosis [4–7]. The most frequent models are the contact burn and the scalding burn [1,5,8–12]. The contact burn is normally induced with the use of a metal bar that is heated in a temperaturecontrolled water bath and applied to the skin for a predetermined amount of time [5,8,9]. In contrast, the scalding model usually employs a template with an aperture through which part of the body is immersed in a water bath with a controlled temperature for a specific period of time [1,10–12]. We devised a refined temperature-controlled apparatus that can be easily assembled and operated, and that is capable of producing different size burns. Associated with a method of fixation and stabilization of the skin, the apparatus allows pressing down perpendicular to the skin surface while maintaining a constant pressure. The objective of the present study was to describe a technique for inducing a burn of standard and constant extent and depth.
2.
Materials and methods
2.1.
Animals
The study was approved by the Ethics Committee on Animal Research of the Biology Institute Roberto Alcantara Gomes, Rio de Janeiro State University, Brazil (protocol number 056/2012). All procedures rigorously followed current guidelines for animal experimentation [13]. Thirty-six male Wistar rats (275–300 g) were used. The animals were housed in appropriate cages, one animal to a cage, under conditions of controlled temperature and humidity, on a 12 h light/12 h dark cycle. Free access to water and standard laboratory chow was allowed. Two days prior to the burn creation the animal dorsum was shaved with electric clippers and depilated with a commercial depilatory cream (VEET(TM) – Rickitt Benckiser Colombia SA; Cali, Colombia). After a 6-h fast, the animals were anesthetized intramuscularly with 80 mg/kg ketamine (Agener Unia˜o; Embu-Guac¸u, Brazil) and 12 mg/kg xylazine (Agener Unia˜o; Embu-Guac¸u, Brazil). The entire procedure was performed under aseptic conditions. After sedation was achieved, the animal was laid on its side with a 2-cm high Styrofoam board adjacent to it. The skin was stretched and fixed with four hypodermic needles (1.25 cm 26G) (Solidor; Barueri, Brazil) so that that the head of the burn apparatus would not only fit completely but would
543
also permit a complete and perpendicular contact of the weighted apparatus – 1 kg of lead (Fig. 1).
2.2.
Burn apparatus
The head of a high power commercial soldering iron (Weller 80 Watts – Cooper Hand Tools; Sorocaba, Brazil) was removed and replaced with one of three cylindrical aluminum heads of different diameters: 19, 23, or 32 mm. A small hole was drilled 3 mm above the contact surface, and a Type K temperature probe (Minipa; Sa˜o Paulo, Brazil) was inserted and attached to a digital thermometer (Minipa MT-401A; Sa˜o Paulo, Brazil) (Fig. 2). Soldering iron works as a resistance. The insertion of a variable autotransformer (JNG TDGC2 0,5KvA; Yueqing, China) between the power outlet and the soldering iron allowed varying the output voltage for a steady AC input voltage. This permits the regulation of the voltage to the soldering iron, leading to a precise control of its temperature. Since the variable autotransformer has an analog dial, we used a digital voltmeter (Minipa ET-1002; Sa˜o Paulo, Brazil) to determine the exact output. During the pretest phase, we determined that to obtain the expected temperatures – 60 8C, 70 8C, and 80 8C – it was necessary to set the output on the voltage autotransformer to 55 V, 60 V, and 65 V, respectively, while the time needed to stabilize the temperature was 20 min.
2.3.
Burns
For the present study we opted for the aluminum cylinder head measuring 23 mm in diameter. Using Meeh’s formula (A = 10 W2/3, where: A = area in cm2, 10 is a constant and W = weight in grams) to calculate the surface area of the animal, the burn represented about 1% of the total surface area [14]. The device was heated to 60 8C, 70 8C, or 80 8C. When the cylinder reached the desired temperature, we waited 5 s for stabilization before applying it to the skin for 10 s. We used 12 animals for each temperature and it was necessary to wait 3 min between animals for the cylinder to recover the desired temperature.
Fig. 1 – The burn apparatus: A – digital voltmeter; B – variable autotransformer; C – aluminum cylindrical heads of different diameters; D – high power commercial soldering iron; E – digital thermometer; F – close up of the type K temperature probe that was inserted into the soldering head.
544
burns 41 (2015) 542–547
Fig. 2 – Animal in position to be submitted to a burn – the skin is held by pins on a Styrofoam board covered with a sterile field: A – top view; B – top view showing complete fit of the soldering head; C – lateral view; D – complete fit of the soldering head.
After the burn, the area was left uncovered and the animals were returned to individual cages. After 3 days, half the animals (6 animals per group) were killed and the burned area was removed with a surrounding margin of healthy skin in order to determine the depth of the burn. The other half of the animals was followed until complete re-epithelialization was achieved.
2.4.
using the Pannoramic Viewer (3DHISTECH Kft.; Budapest, Hungary); all slides were first scanned with a Pannoramic MIDI digital slide scanner (3DHISTECH Kft.; Budapest, Hungary). Results are presented in 2 forms: the average total depth of the burn (including the muscle layer) – in mm – and the total percentage of dermis involved. To calculate the percentage of the dermis involved, burn depth was multiplied by 100 and divided by the dermis depth.
Macroscopic analyses 2.6.
To evaluate wound re-epithelialization, a transparent plastic sheet was placed over the wound and its margins were traced every 7 days until complete re-epithelialization. After digitalization, the wound area was measured using the ImageJ 1.47 v software (National Institutes of Health; Bethesda, MD, USA). The wound area was measured and photographed soon after the burn and every 7 days until complete re-epithelialization without scab removal. Data are reported as percentage of the initial burn area.
2.5.
Statistical analysis
All results are reported as mean standard deviation. Significant differences among groups were evaluated using Mann–Whitney. A difference of p < 0.05 was considered to be statistically different. All analyses were performed using the GraphPad Prisma V5.0 software (Graphpad Software Inc; La Jolla, CA, USA).
3.
Results
Histological evaluation
The lesions were excised with surrounding healthy skin and fixed in buffered 10% formaldehyde. The tissue was processed with increasing concentrations of alcohol and xylol, embedded in paraffin, and cut into 5 mm-thick slices. Slides prepared from these slices were stained with hematoxylin-and-eosin and analyzed by light microscopy. The examiner was blinded to the groups he was measuring. Criteria to evaluate burn included cellular necrosis, vascular occlusion and collagen injury [15]. The depth of the burn and the total thickness of the dermis were measured at 5 different points in the wound, from right to left at the 1/4, 3/8, 1/2, 5/8, and 3/4 distance points,
We did not observe deaths among the animals. The burns produced were homogeneous and round, and their margins were visible since the moment when the burn was created. Three days after the burn, the burned area was still clearly visible and homogeneous in all groups (Fig. 3a–c). Histological observation showed that the burned skin presented epidermal necrosis, diffuse perivascular infiltration, and collagen degeneration, while the healthy skin had a characteristic histologic appearance. Animals that were exposed to 60 8C developed a superficial second-degree burn (damage limited to the upper third of the dermis) (Fig. 3d), the animals submitted to 70 8C presented a deep second-degree
burns 41 (2015) 542–547
545
Fig. 3 – Macroscopic (A, B, and C) and microscopic (D, E, and F) aspects of the burn lesion at different temperatures: A – 60 8C burn; B – 70 8C burn; C – 80 8C burn; D – 60 8C burn; central bar showing lesion of the epidermis and of the upper part of the dermis; superficial second degree burn; E – 70 8C burn; central bar showing lesion of almost all dermis; deep second degree burn; F – 80 8C burn; central bar showing full lesion of the dermis and muscular layer; third degree burn.
burn (damage involving the lower two thirds of the dermis) (Fig. 3e), and in the 80 8C group, a third degree-burn was observed, with involvement of all skin layers, including the superficial portion of the intradermal muscle layer ( panniculus carnosus) (Fig. 3f). Burn depth, reported as total depth or percentage of dermis involved, differed significantly between groups. The mean total depth was 350.2 48.32 mm for 60 8C, 847.2 116.0 mm for 70 8C, and 1915 270.3 mm for 80 8C (P < 0.05). This represented the involvement of 28%, 72%, and 100% of the dermis for 60 8C, 70 8C, and 80 8C, respectively (Fig. 4). We observed a difference in the time needed for wound re-epithelialization between the temperatures used to create it. For the different temperatures (60 8C, 70 8C, and 80 8C) it took 21, 35, and 49 days for full closure, respectively (Fig. 5). A difference of 2 weeks was observed between each temperature for full closure of the wound.
4.
Discussion
Initially, it is important to explain that in the model we designed we took into consideration a couple of important facts: depilation is mandatory because hair may hinder the achievement of a uniform burn [7,8] and the demarcation of necrosis was stable by the third day [5,7], this being the reason why the animals were killed by that time. Burns can be classified into first, second and third degrees, with first-degree burns being limited to the epidermis, seconddegree burns involving the epidermis and part of the dermis, and third-degree burns destroying the epidermis and all dermis [6,16]. We were able to create second- and third-degree burns in the animals depending on which temperature was employed. Although we used only the aluminum cylinder
546
burns 41 (2015) 542–547
Fig. 4 – Evaluation of burn depth. A – Total burn depth in mm. B – Percentage of dermis burned. Data presented as mean W standard deviation * p < 0.05.
head measuring 23 mm in diameter, it is also possible to vary the total burn area by changing the head of the apparatus. Scalding models are cumbersome, some requiring up to three persons to produce each burn [10] and may also pose a threat to the operators involved [5]. In these cases, it is imperative to create a perfect water seal, so that the only contact with the predetermined exposed area of the template is the heated liquid, without any dripping or leakage that might lead to an increase in the total burn area [12,17]. In the contact burn model, an aluminum bar is heated by immersion in a controlled temperature liquid bath but there is no accurate way to know the exact temperature of the bar or how long it must remain immersed for its temperature to stabilize [4]. In addition, the vapor on the bar surface can create ‘‘cold spots’’ that lead to irregular contact and ultimately affect the burn depth [18]. Another important issue is that the applied pressure is unpredictable [4]. We employed an aluminum bar heated by a controlled electric current with a temperature probe inserted in the head for accurate monitoring and, since no water was involved, the risk of spilling, dripping, formation of cold spots or other risks to the investigator were eliminated.
Fig. 5 – Percentage of original wound area (data presented as mean W standard deviation). For a 60 8C burn, complete re-epithelialization occurred on day 21, for a 70 8C burn on day 35 and for a 80 8C burn on day 49.
On day 3, histological analysis of the burn depth showed direct and constant results associated with the temperatures, with higher temperatures producing deeper burns. In relation to dermis involvement, the temperature of 60 8C produced a superficial second degree burn affecting 28% of dermis thickness, 70 8C produced a deep second degree burn affecting 72% of dermis thickness, and 80 8C produced a third degree burn affecting 100% of dermis thickness. Time for wound re-epithelialization was also affected by the different temperatures, with 14 days of difference between temperatures and with deeper burns taking longer to heal. Although some authors prefer scalding methods to a contact-based method, the latter permits a better therapeutic opportunity since it causes a second- rather than a thirddegree burn, but this premise is based on a study that compared the methods at very different temperatures (80 8C vs. 170 8C) [9]. It is clear that the depth of a burn is directly influenced by temperature, so it is not accurate to compare burn methods using different temperatures. Scald burns take longer than contact burns to determine their final depth because the immediate necrotic layer formed by the contact burn acts as a protective barrier against heat transfer to the deep vascular plexus. When the plexus is affected, we have not only the injury due to the heat source, but also from delayed vascular damage [9]. In contact burn models, it is imperative to have a large enough area of skin to permit a full contact [5,7] with uniform pressure [19] since pressure has a direct influence on burn depth. In the present model, these issues were addressed by positioning the animal on its side, with fixation of the excess of skin located on the lateral wall of the thorax to a solid base (Styrofoam) associated with the addition of weight to the apparatus (1 kg) to allow a uniform pressure contact. Without uniform pressure it is impossible to obtain a uniform depth [18,19], so the association of the apparatus and the described technique creates pressure on the animal that is operator-independent and ensures uniform burns. As described, this model is not totally risk proof but involves a significant risk reduction because of easy manipulation and the need for a reduced number of people to fully execute the experiment.
burns 41 (2015) 542–547
This model permits a precise burn with a constant depth, so it seems ideal for comparing treatments and can even be used in a split model (with control on one side and treatment on the other). The influence on total depth, time for healing, and effect on tissue viability can be assessed.
[2]
[3]
5.
Conclusion
The standardization and reproducibility of experimental models are indispensable for all scientific research and this can only be achieved with a detailed description of the instruments and the method used, along with their advantages and limitations. Although we gave a detailed description of how to manufacture the apparatus, it is important to highlight that when any apparatus is built from multiple industrial parts, which may lack fine precision, the final product must be tested in order to insure that the selected settings correspond to the desired parameters. This simple, safe, fast, and low-cost method produces reliable and consistent burn wounds that can range from full to partial thickness burns of adjustable diameters. The control of most of the variables with this method provides an extremely useful experimental burn model.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Funding [11]
The authors have no financial interest to declare in relation to the content of this article. This work was partially supported by FAPERJ – Fundac¸a˜o de Amparo a Pesquisa Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro and CNPq – Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (grant number E-26-111.991/2012).
[12] [13] [14]
[15]
Author’s contribution NGV, AMAC and RGM designed the experiment. AMAC and NGV conducted the experiment. NGV, AMAC and RGM analyzed data. NGV wrote manuscript. AMAC and RGM revised manuscript.
references
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[18]
[19] [1] Latif R, Thyagarajan SP, Subramanian S, Ananthan S, Sridhar G, Udayasankar K, et al. Development of a simple
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burn mouse model and its evaluation to study the efficacy of soframycin against Pseudomonas aeruginosa infected burns. Indian J Med Microbiol 1992;10(1):23–8. Burn incidence and treatment in the United States: 2012 fact sheet; 2012, Available from: http://www.ameriburn. org/resources_factsheet.php. Munster AM. Measurements of quality of life: then and now. Burns 1999;25(1):25–8. Singer JA, Taira BR, Anderson R, McClain SA, Rosenberg L. Does pressure matter in creating burns in a porcine model? J Burn Care Res 2010;31(4):646–51. Singer AJ, Berruti L, Thode Jr HC, McClain SA. Standardized burn model using a multiparametric histologic analysis of burn depth. Acad Emerg Med 2000;7(1):1–6. Wang X, Kimble R. A review on porcine burn and scar models and their relevance to humans. Wound Pract Res 2010;18(1):41–9. Knabl JS, Bayer GS, Bauer WA, Schwendenwein I, Dado PF, Kucher C, et al. Controlled partial skin thickness burns: an animal model for studies of burnwound progression. Burns 1999;25(3):229–35. Kaufman T, Lusthaus SN, Sagher U, Wexler MR. Deep partial skin thickness burns: a reproducible animal model to study burn wound healing. Burns 1990;16(1): 13–6. Brans TA, Dutrieux RP, Hoekstra MJ, Kreis RW, du Pont JS. Histopathological evaluation of scalds and contact burns in the pig model. Burns 1994;20(Suppl. 1):S48–51. Pfurtscheller K, Petnehazy T, Goessler W, WiedersteinGrasser I, Bubalo V, Trop M. Innovative scald burn model and long-term dressing protector for studies in rats. J Trauma Acute Care Surg 2013;74(3):932–5. Davis JW, Hansbrough JF. Use of overhead hooks in burn surgery. J Burn Care Rehabil 1988;9(5):492. Walker HL, Mason Jr AD. A standard animal burn. J Trauma 1968;8(6):1049–51. Marques RG, Morales MM, Petroianu A. Brazilian law for scientific use of animals. Acta Cir Bras 2009;24(1):69–74. Meyer TN, Silva AL. A standard burn model using rats. Acta Cir Bras 1999;14(Oct-Dec (4)) [serial online] Available from: URL: http://www.scielo.br/acb. Hirth DA, Singer AJ, Clark RA, McClain SA. Histopathologic staining of low temperature cutaneous burns: comparing biomarkers of epithelial and vascular injury reveals utility of HMGB1 and hematoxylin phloxine saffron. Wound Repair Regen 2012;20(6):918–27. Singh V, Devgan L, Bhat S, Milner SM. The pathogenesis of burn wound conversion. Ann Plast Surg 2007;59(1):109–15. Dinar S, Sen C, Unal C, Agir H, Iscen D. A new material for the standard burn model: Allevyn adhesive. Plast Reconstr Surg 2006;117(2):717–8. Gaines C, Poranki D, Du W, Clark RA, Van Dyke M. Development of a porcine deep partial thickness burn model. Burns 2013;39(2):311–9. Heredero FXS, Hammann C, Obispo Martins JM, Rodrigues Arias C, Coca Menchero S. Experimental burn models. Ann Burns Fire Disasters 1996;9(2):96–7.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/burns
A new model for the standardization of experimental burn wounds Neil G. Venter a,*, Andre´a Monte-Alto-Costa b, Ruy G. Marques a a
Department of Surgery, Rio de Janeiro State University, Boulevard Vinte e Oito de Setembro, 77, Vila Isabel, Rio de Janeiro, RJ 20551-030, Brazil b Department of Histology and Embryology, Rio de Janeiro State University, Av. Marechal Rondon, 381/HLA, Sa˜o Francisco Xavier, Rio de Janeiro, RJ 20950-003, Brazil
article info
abstract
Article history:
Background: Burns are common and recurrent events treated by physicians on a daily basis
Accepted 7 August 2014
at most emergency rooms around the world. There is a constant need to understand the physiopathology of burns, so as to minimize their devastating results. The objective of the
Keywords:
present report is to describe a burn apparatus in association with an innovative method of
Experimental model
animal fixation, as to produce burns of varying sizes and depths.
Burn
Methods: Rats were subjected to burns of 60 8C, 70 8C, and 80 8C for 10 s and after 3 days half of the rats in each group were killed and the resulting lesions were analyzed using histological techniques. In the other half of the rats the wound was measured weakly until complete re-epithelialization. Results: All burns were easily visible and the histological feature for the 60 8C burn was a superficial second-degree burn (28% of the dermis), for 70 8C we observed a deep seconddegree burn (72% of the dermis), and in the 80 8C group, a third degree-burn was present (100% of the dermis). Conclusions: This is a safe, reliable, easy to construct and use model that has the ability to produce a regular and uniform reproducible burn due to precise temperature control associated with standardized animal positioning. # 2014 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Burns are among the most common injuries in modern life [1]. In the United States alone, every year 450,000 patients receive medical treatment related to burns, with over 40,000 hospital admissions and 3500 deaths due to burns [2].
Initial studies concerning burns were focused on improving the overall survival rate. These works provided a better understanding of the physiopathology of burns and led to enhanced resuscitation techniques that drastically reduced the number of deaths of burn victims during the early phases of injury. In a second phase, the focus of investigation shifted toward minimizing morbidity and improving the quality of life.
* Corresponding author at: Av. Ayrton Senna, 170/2008 Barra da Tijuca, Rio de Janeiro, RJ 22793-000, Brazil. Tel.: +55 21 997797500; fax: +55 21 37964417. E-mail addresses: [email protected] (N.G. Venter), [email protected] (A. Monte-Alto-Costa), [email protected] (R.G. Marques). Abbreviations: C, Celsius; V, Volts. http://dx.doi.org/10.1016/j.burns.2014.08.002 0305-4179/# 2014 Elsevier Ltd and ISBI. All rights reserved.
burns 41 (2015) 542–547
Nowadays, one of the primary approaches concerns scar quality, especially when the burn does not affect a large body surface [3]. Therefore, in experimental models, the burn areas must be small enough not to have a systemic repercussion, but yet large enough to permit adequate observation and sampling. An experimental model is essential to test therapies before clinical use. In order to evaluate the effectiveness of burn wound treatment, however, it is extremely important to be able to create predictable and uniform burns, where most of the confounding variables can be eliminated. Burn depth, defined by three different elements – temperature, time of exposure, and contact pressure – is the primary determinant of prognosis [4–7]. The most frequent models are the contact burn and the scalding burn [1,5,8–12]. The contact burn is normally induced with the use of a metal bar that is heated in a temperaturecontrolled water bath and applied to the skin for a predetermined amount of time [5,8,9]. In contrast, the scalding model usually employs a template with an aperture through which part of the body is immersed in a water bath with a controlled temperature for a specific period of time [1,10–12]. We devised a refined temperature-controlled apparatus that can be easily assembled and operated, and that is capable of producing different size burns. Associated with a method of fixation and stabilization of the skin, the apparatus allows pressing down perpendicular to the skin surface while maintaining a constant pressure. The objective of the present study was to describe a technique for inducing a burn of standard and constant extent and depth.
2.
Materials and methods
2.1.
Animals
The study was approved by the Ethics Committee on Animal Research of the Biology Institute Roberto Alcantara Gomes, Rio de Janeiro State University, Brazil (protocol number 056/2012). All procedures rigorously followed current guidelines for animal experimentation [13]. Thirty-six male Wistar rats (275–300 g) were used. The animals were housed in appropriate cages, one animal to a cage, under conditions of controlled temperature and humidity, on a 12 h light/12 h dark cycle. Free access to water and standard laboratory chow was allowed. Two days prior to the burn creation the animal dorsum was shaved with electric clippers and depilated with a commercial depilatory cream (VEET(TM) – Rickitt Benckiser Colombia SA; Cali, Colombia). After a 6-h fast, the animals were anesthetized intramuscularly with 80 mg/kg ketamine (Agener Unia˜o; Embu-Guac¸u, Brazil) and 12 mg/kg xylazine (Agener Unia˜o; Embu-Guac¸u, Brazil). The entire procedure was performed under aseptic conditions. After sedation was achieved, the animal was laid on its side with a 2-cm high Styrofoam board adjacent to it. The skin was stretched and fixed with four hypodermic needles (1.25 cm 26G) (Solidor; Barueri, Brazil) so that that the head of the burn apparatus would not only fit completely but would
543
also permit a complete and perpendicular contact of the weighted apparatus – 1 kg of lead (Fig. 1).
2.2.
Burn apparatus
The head of a high power commercial soldering iron (Weller 80 Watts – Cooper Hand Tools; Sorocaba, Brazil) was removed and replaced with one of three cylindrical aluminum heads of different diameters: 19, 23, or 32 mm. A small hole was drilled 3 mm above the contact surface, and a Type K temperature probe (Minipa; Sa˜o Paulo, Brazil) was inserted and attached to a digital thermometer (Minipa MT-401A; Sa˜o Paulo, Brazil) (Fig. 2). Soldering iron works as a resistance. The insertion of a variable autotransformer (JNG TDGC2 0,5KvA; Yueqing, China) between the power outlet and the soldering iron allowed varying the output voltage for a steady AC input voltage. This permits the regulation of the voltage to the soldering iron, leading to a precise control of its temperature. Since the variable autotransformer has an analog dial, we used a digital voltmeter (Minipa ET-1002; Sa˜o Paulo, Brazil) to determine the exact output. During the pretest phase, we determined that to obtain the expected temperatures – 60 8C, 70 8C, and 80 8C – it was necessary to set the output on the voltage autotransformer to 55 V, 60 V, and 65 V, respectively, while the time needed to stabilize the temperature was 20 min.
2.3.
Burns
For the present study we opted for the aluminum cylinder head measuring 23 mm in diameter. Using Meeh’s formula (A = 10 W2/3, where: A = area in cm2, 10 is a constant and W = weight in grams) to calculate the surface area of the animal, the burn represented about 1% of the total surface area [14]. The device was heated to 60 8C, 70 8C, or 80 8C. When the cylinder reached the desired temperature, we waited 5 s for stabilization before applying it to the skin for 10 s. We used 12 animals for each temperature and it was necessary to wait 3 min between animals for the cylinder to recover the desired temperature.
Fig. 1 – The burn apparatus: A – digital voltmeter; B – variable autotransformer; C – aluminum cylindrical heads of different diameters; D – high power commercial soldering iron; E – digital thermometer; F – close up of the type K temperature probe that was inserted into the soldering head.
544
burns 41 (2015) 542–547
Fig. 2 – Animal in position to be submitted to a burn – the skin is held by pins on a Styrofoam board covered with a sterile field: A – top view; B – top view showing complete fit of the soldering head; C – lateral view; D – complete fit of the soldering head.
After the burn, the area was left uncovered and the animals were returned to individual cages. After 3 days, half the animals (6 animals per group) were killed and the burned area was removed with a surrounding margin of healthy skin in order to determine the depth of the burn. The other half of the animals was followed until complete re-epithelialization was achieved.
2.4.
using the Pannoramic Viewer (3DHISTECH Kft.; Budapest, Hungary); all slides were first scanned with a Pannoramic MIDI digital slide scanner (3DHISTECH Kft.; Budapest, Hungary). Results are presented in 2 forms: the average total depth of the burn (including the muscle layer) – in mm – and the total percentage of dermis involved. To calculate the percentage of the dermis involved, burn depth was multiplied by 100 and divided by the dermis depth.
Macroscopic analyses 2.6.
To evaluate wound re-epithelialization, a transparent plastic sheet was placed over the wound and its margins were traced every 7 days until complete re-epithelialization. After digitalization, the wound area was measured using the ImageJ 1.47 v software (National Institutes of Health; Bethesda, MD, USA). The wound area was measured and photographed soon after the burn and every 7 days until complete re-epithelialization without scab removal. Data are reported as percentage of the initial burn area.
2.5.
Statistical analysis
All results are reported as mean standard deviation. Significant differences among groups were evaluated using Mann–Whitney. A difference of p < 0.05 was considered to be statistically different. All analyses were performed using the GraphPad Prisma V5.0 software (Graphpad Software Inc; La Jolla, CA, USA).
3.
Results
Histological evaluation
The lesions were excised with surrounding healthy skin and fixed in buffered 10% formaldehyde. The tissue was processed with increasing concentrations of alcohol and xylol, embedded in paraffin, and cut into 5 mm-thick slices. Slides prepared from these slices were stained with hematoxylin-and-eosin and analyzed by light microscopy. The examiner was blinded to the groups he was measuring. Criteria to evaluate burn included cellular necrosis, vascular occlusion and collagen injury [15]. The depth of the burn and the total thickness of the dermis were measured at 5 different points in the wound, from right to left at the 1/4, 3/8, 1/2, 5/8, and 3/4 distance points,
We did not observe deaths among the animals. The burns produced were homogeneous and round, and their margins were visible since the moment when the burn was created. Three days after the burn, the burned area was still clearly visible and homogeneous in all groups (Fig. 3a–c). Histological observation showed that the burned skin presented epidermal necrosis, diffuse perivascular infiltration, and collagen degeneration, while the healthy skin had a characteristic histologic appearance. Animals that were exposed to 60 8C developed a superficial second-degree burn (damage limited to the upper third of the dermis) (Fig. 3d), the animals submitted to 70 8C presented a deep second-degree
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Fig. 3 – Macroscopic (A, B, and C) and microscopic (D, E, and F) aspects of the burn lesion at different temperatures: A – 60 8C burn; B – 70 8C burn; C – 80 8C burn; D – 60 8C burn; central bar showing lesion of the epidermis and of the upper part of the dermis; superficial second degree burn; E – 70 8C burn; central bar showing lesion of almost all dermis; deep second degree burn; F – 80 8C burn; central bar showing full lesion of the dermis and muscular layer; third degree burn.
burn (damage involving the lower two thirds of the dermis) (Fig. 3e), and in the 80 8C group, a third degree-burn was observed, with involvement of all skin layers, including the superficial portion of the intradermal muscle layer ( panniculus carnosus) (Fig. 3f). Burn depth, reported as total depth or percentage of dermis involved, differed significantly between groups. The mean total depth was 350.2 48.32 mm for 60 8C, 847.2 116.0 mm for 70 8C, and 1915 270.3 mm for 80 8C (P < 0.05). This represented the involvement of 28%, 72%, and 100% of the dermis for 60 8C, 70 8C, and 80 8C, respectively (Fig. 4). We observed a difference in the time needed for wound re-epithelialization between the temperatures used to create it. For the different temperatures (60 8C, 70 8C, and 80 8C) it took 21, 35, and 49 days for full closure, respectively (Fig. 5). A difference of 2 weeks was observed between each temperature for full closure of the wound.
4.
Discussion
Initially, it is important to explain that in the model we designed we took into consideration a couple of important facts: depilation is mandatory because hair may hinder the achievement of a uniform burn [7,8] and the demarcation of necrosis was stable by the third day [5,7], this being the reason why the animals were killed by that time. Burns can be classified into first, second and third degrees, with first-degree burns being limited to the epidermis, seconddegree burns involving the epidermis and part of the dermis, and third-degree burns destroying the epidermis and all dermis [6,16]. We were able to create second- and third-degree burns in the animals depending on which temperature was employed. Although we used only the aluminum cylinder
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Fig. 4 – Evaluation of burn depth. A – Total burn depth in mm. B – Percentage of dermis burned. Data presented as mean W standard deviation * p < 0.05.
head measuring 23 mm in diameter, it is also possible to vary the total burn area by changing the head of the apparatus. Scalding models are cumbersome, some requiring up to three persons to produce each burn [10] and may also pose a threat to the operators involved [5]. In these cases, it is imperative to create a perfect water seal, so that the only contact with the predetermined exposed area of the template is the heated liquid, without any dripping or leakage that might lead to an increase in the total burn area [12,17]. In the contact burn model, an aluminum bar is heated by immersion in a controlled temperature liquid bath but there is no accurate way to know the exact temperature of the bar or how long it must remain immersed for its temperature to stabilize [4]. In addition, the vapor on the bar surface can create ‘‘cold spots’’ that lead to irregular contact and ultimately affect the burn depth [18]. Another important issue is that the applied pressure is unpredictable [4]. We employed an aluminum bar heated by a controlled electric current with a temperature probe inserted in the head for accurate monitoring and, since no water was involved, the risk of spilling, dripping, formation of cold spots or other risks to the investigator were eliminated.
Fig. 5 – Percentage of original wound area (data presented as mean W standard deviation). For a 60 8C burn, complete re-epithelialization occurred on day 21, for a 70 8C burn on day 35 and for a 80 8C burn on day 49.
On day 3, histological analysis of the burn depth showed direct and constant results associated with the temperatures, with higher temperatures producing deeper burns. In relation to dermis involvement, the temperature of 60 8C produced a superficial second degree burn affecting 28% of dermis thickness, 70 8C produced a deep second degree burn affecting 72% of dermis thickness, and 80 8C produced a third degree burn affecting 100% of dermis thickness. Time for wound re-epithelialization was also affected by the different temperatures, with 14 days of difference between temperatures and with deeper burns taking longer to heal. Although some authors prefer scalding methods to a contact-based method, the latter permits a better therapeutic opportunity since it causes a second- rather than a thirddegree burn, but this premise is based on a study that compared the methods at very different temperatures (80 8C vs. 170 8C) [9]. It is clear that the depth of a burn is directly influenced by temperature, so it is not accurate to compare burn methods using different temperatures. Scald burns take longer than contact burns to determine their final depth because the immediate necrotic layer formed by the contact burn acts as a protective barrier against heat transfer to the deep vascular plexus. When the plexus is affected, we have not only the injury due to the heat source, but also from delayed vascular damage [9]. In contact burn models, it is imperative to have a large enough area of skin to permit a full contact [5,7] with uniform pressure [19] since pressure has a direct influence on burn depth. In the present model, these issues were addressed by positioning the animal on its side, with fixation of the excess of skin located on the lateral wall of the thorax to a solid base (Styrofoam) associated with the addition of weight to the apparatus (1 kg) to allow a uniform pressure contact. Without uniform pressure it is impossible to obtain a uniform depth [18,19], so the association of the apparatus and the described technique creates pressure on the animal that is operator-independent and ensures uniform burns. As described, this model is not totally risk proof but involves a significant risk reduction because of easy manipulation and the need for a reduced number of people to fully execute the experiment.
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This model permits a precise burn with a constant depth, so it seems ideal for comparing treatments and can even be used in a split model (with control on one side and treatment on the other). The influence on total depth, time for healing, and effect on tissue viability can be assessed.
[2]
[3]
5.
Conclusion
The standardization and reproducibility of experimental models are indispensable for all scientific research and this can only be achieved with a detailed description of the instruments and the method used, along with their advantages and limitations. Although we gave a detailed description of how to manufacture the apparatus, it is important to highlight that when any apparatus is built from multiple industrial parts, which may lack fine precision, the final product must be tested in order to insure that the selected settings correspond to the desired parameters. This simple, safe, fast, and low-cost method produces reliable and consistent burn wounds that can range from full to partial thickness burns of adjustable diameters. The control of most of the variables with this method provides an extremely useful experimental burn model.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Funding [11]
The authors have no financial interest to declare in relation to the content of this article. This work was partially supported by FAPERJ – Fundac¸a˜o de Amparo a Pesquisa Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro and CNPq – Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (grant number E-26-111.991/2012).
[12] [13] [14]
[15]
Author’s contribution NGV, AMAC and RGM designed the experiment. AMAC and NGV conducted the experiment. NGV, AMAC and RGM analyzed data. NGV wrote manuscript. AMAC and RGM revised manuscript.
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