An optimized animal model for partial and total skin thickness burns studies 1 Um modelo animal aperfeiçoado para estudo de queimaduras superficiais e profundas da pele

PURPOSE: Development of an improved animal model for studying skin burns in rats. METHODS: Twenty-four male Wistar rats were randomly assigned to four groups (n=6): G1-Control, G2T100°C, G3-T150°C and G4-T200°C. Two 10 x 10 mm squares were outlined with a sterile surgical marker on each side and along the vertebral column using a prepared template positioned between the anterior and posterior limbs. G2-G4 rats were subjected to 100°C, 150°C and 200oC thermal burns, respectively. G1 rats served as controls. Burns were inflicted by applying a copper plate connected to an electronic temperature controlling device to the dorsal skin of anesthetized rats. Four burns were produced on each animal (total area: 4 cm2/ animal) leaving about 1 cm of undamaged skin between burn areas. Analgesia was administered during 24 h after burn injury by adding 30 mg codeine phosphate hemihydrate to 500 ml tap water. RESULTS: The application of 100°C and 150oC resulted in partial thickness skin burns with central reepithelialization of the burned area only at 100°C. In G4 group the whole thickness of the skin was injured without central reepithelialization. However, there was marginal reepithelialization in all groups. CONCLUSION: The model studied is inexpensive and easily reproducible, enabling the achievement of controlled burns with partial or total impairment of the skin in experimental animals.


Introduction
Induction of experimental burns in animal models is an essential tool to understand the pathogenesis of skin damage due to burns in humans and for designing new methods of treatment.Animal models have been used to investigate burn wound pathology, local therapy 1 , the influence of systemic drug application on the burn wound and the effect of burn trauma on the entire organism 2 .Inconsistency and irreproducibility are commonly found in experimental studies.Two factors may be related to the controversial and different findings presented in the available literature: burn models are not identical or different modalities of treatment are used by researchers.
The present study was therefore undertaken to determine the optimal experimental model of burns heat-induced by direct conduction in rats, correlating standardized temperatures and lesion depths (partial or total skin thickness burn).

Methods
Approval for experimental use of laboratory animals was obtained on September, 2010 (Protocol #37/ The study was designed to minimize the number of animals required for the experiments.The animals were housed in polypropylene cages at ambient temperature of 24 o C on a 12 h light-dark cycle.

Study design
Twenty-four male Wistar rats were randomly assigned to four groups (n=6): G1-Control, G2-T100°C, G3-T150°C and G4-T200°C.Two 10 x 10 mm squares were outlined with a sterile surgical marker on each side and along the vertebral column using a prepared template (an X-ray film with a 10 x 10 mm grid) positioned between the anterior and posterior limbs.G2-G4 rats were subjected to 100°C, 150°C and 200°C thermal burns, respectively.G1 rats served as controls.Immediately before inflicting the burn, the area was shaved with a standard electric shaving machine to obtain a smooth surface and hairless skin.The dorsal skin was surgically prepared with successive applications of 2% chlorhexidine scrub.A previously described model 3,4 was used with some modifications.The original tip of an ordinary 40W soldering iron was replaced with tip and square 10 x 10mm copper plate.An electronic temperature controller with a thermocoupletype feedback sensor was connected 2 mm above the plate tip in order to allow precise optimal temperature monitoring at the tip of the instrument.Additionally, a digital multimeter with a K type thermocouple was fixed to the copper plate to assure real time optimal control of the temperature applied to the skin (surface counter).The desired stamp temperature (100°C, 150°C and 200°C) was reached 5 min after switching on the electric current.The device was positioned vertically under its own weight (85g) and applied to each skin burn site during 9 seconds to inflict the burns as outlined.Immediately after each burn injury, the respective wound was cooled off during 1 min with gauze embedded in isotonic saline at 22°C, as described elsewhere 5 .The plate produced a burn area of approximately 1 cm 2 / wound.Four burns were produced on each animal (4 cm 2 /animal) leaving approximately 1 cm intact skin between burn areas.Following conclusion of the procedure, the animals were returned to their individual cages for recovery with free access to rat chow and tap water.Analgesia was administered during 24h after burn injury by adding 30 mg codeine phosphate hemihydrate to 500 ml tap water.

Anesthesia
Anesthesia was induced with an intramuscular injection of ketamine hydrochloride (90 mg/kg) and xylazine (10 mg/kg) to all rats.

Macroscopic analysis
Anesthetized rats were photographed in the seventh day post-burn under anesthesia and standard lighting conditions using a tripod-mounted digital camera (Sony DSC-W35, 7.2 mega pixels) with a constant 1.0 x zoom at 35 cm distance from the dorsum of the animal.The Images (format TIFF, 512 x 384 pixels) were calibrated and the wound surface area (mm 2 ) and perimeter (mm) were calculated by computerized planimetry using the software Image Tool 3.0 (University of Texas Health Center at San Antonio).

Histopathology
At the end of the experiments all animals were killed by an overdose of anesthetics (Ketamine+xylazine).Tissue samples were collected from the dorsum with a surgical blade, removing skin fragments (20 mm x 5 mm), including the central scar, adjacent healthy tissue and underlying panniculus carnosus muscle.Tissue samples were fixed in formalin for 24 hours before being transferred to 70% ethanol solution.Further processing included paraffin embedding and sectioning, to generate 5-µm-thick tissue coronal sections to be mounted on glass slides.The slides were stained using hematoxylin and eosin.The extent of skin damage was assessed by a blind pathologist using a light microscope (Olympus, X100).

Statistical analysis
Graphpad Prism 5.0 (GraphPad Software, www.graphpad.com)was used for statistical analysis and graphics design.Data from images (wound surface area and perimeter) were tested for distribution (Kolmorogov-Smirnov test).Results were expressed as mean+SD.Groups were compared at different temperature (100 °C, 150 °C and 200 °C) using Student's t test for unpaired variables.The level of statistical significance was set at 5%.Histopathology findings were tabulated for descriptive analysis.

Results
No infection or mortality was observed during the experiments and no systemic complications were clinically detectable.

Microscopic analysis
Scars were present in all wounds along incipient marginal reepithelization (Figure 4, images E, G and J).Incipient central reepithelialization was present in G2 only (Figure 4, image F).The dermis presented obliterated vessels and partially damaged pilosebaceous units in groups G2 and G3 (Figure 4, images C, E and H).Total dermis and muscle damage were found in G4 rats (Figure 4, images J and L).Musculature layers were preserved in G2 and G3 rats (Figure 4, images D and I, respectively).Table 1 depicts the Presence/absence of skin structures and reepithelialization in G2-G4 rats.An optimized animal model for partial and total skin thickness burns studies Acta Cirúrgica Brasileira -Vol.26 (Suppl.1) 2011 -41 In order to achieve consistent reproducibility, the stamp pressure should be identical in all lesions and positions to ensure maximal skin contact.A major advantage of the hand-held stamp using the device weight to induce the burn lesion is that it remains constant throughout the procedure and is not related to the pressure exerted by the researcher that can present some changes due to the difficulty of figuring out the intensity of pressure that should be applied to the skin of the animal.

Conclusion
The model studied is inexpensive and easily reproducible, enabling the achievement of controlled burns with partial or total impairment of the skin in experimental animals.

Discussion
Over the past decade much has been discovered concerning the pathophysiology and forms of treatment of burn lesions.Different burn models have been presented.Methods for producing experimental burns included boiling water 6 , heated brass bar 7 , heated (130°C) aluminum plate 8 , a constant-temperature hot plate (170ªC) placed over stainless steel rods 9 and scalding over 45% of the body surface 10 .
In the present study, skin burns were inflicted by transference of energy (heat) using direct conduction (Copper/skin) utilizing different increasing temperatures (100°C, 150°C and 200°C).Thermal energy transfer by conduction from a solid structure to the skin surface does not depend on the pressure employed, but mainly on the temperature gradient between the skin, the solid structure and the distance between those elements 11 .To the best of our knowledge, this is it the fist work where a copper template is used to induce thermal burns.Copper thermal conductivity is more efficient than other metals used by researchers 1,7,12 .
The results clearly demonstrated that this experimental model provided excellent contrast between normal and burned tissue at different temperatures.Thus, the highest temperature used (200°C) producing lesions in all skin layers with damaged pilosebaceous units resulting in poor central reepithelialization, making being this method a good choice to evaluate drug effects in severe burnings.
Moreover, it was possible to obtain partial skin thickness burn with lesser temperatures (100°C and 150°C).However, burning at 150°C was more deeper than 100°C and the central reepithelialization occurred only when the plate was heated to 100°C.The partial-thickness burn model (using 100°C and 150°C) in this study spares some epithelial elements within the dermis, allowing for spontaneous regeneration (reepithelialization) via proliferation and migration of epithelial cells from dermal appendages remaining at the wound site and from the wound margins [13][14] .Practically, it implies that a deep partial skin thickness burn should not be converted into a full skin thickness injury.

FIGURE 1 -
FIGURE 1 -Macroscopic appearance of burns on post-burn day 7.

TABLE 1 -
Presence/absence of skin structures and recovery (reepithelialization) in burned rats