burns are tissue lesions from thermal origin for exposure to flames , hot surfaces and liquids , extreme cold , chemicals , radiation , or friction .
even with improved prognosis   and progress in the use of biological skin substitutes , burns are an important cause of mortality .
burns are classified depending on the lesion severity into superficial or first degree , when lesion is restricted to the epidermis or skin causing redness ; partial thickness or second degree that can be superficial when reaching the epidermis and superficial dermis , showing hypersensitivity and pain , or deep when it extends to the deepest layer of the dermis and may have reduced sensitivity with red and/or white coloration of the tissue ; full - thickness or third degree when lesion involves the subcutaneous layer , with no sensitivity and white coloring .
the use of animals as experimental models in different areas of biological research was encouraged by claude bernard , who around 1865 , described in his paper entitled  introduction to the study of experimental medicine  the use of animals as a model for study and transposition into human physiology .
there are literature reports on the use of rabbits , pigs , dogs , rats , and mice   as models in the study of burns .
the healing of skin lesions induces the burn - injured tissue inflammation , edema , and hypertrophic and unsightly scars .
thus , the choice of a topical agent or the type of coverage to be used in treating burns should be conducted based on the assessment of lesion characteristics and evidence reported in the specific literature .
these products must have features such as antimicrobial or bacteriostatic activity , absence of toxicity and hypersensitivity , compliance , reduced healing time , and cost / benefit .
however , many of the methods used in healing injuries caused by burns are controversial . in this context , the objective of this study was to establish an experimental protocol for induction of deep second - degree thermal lesions in wistar male rats to obtain clinical and histopathologic data that will facilitate understanding of results concerning the evolution of the healing action of topical therapeutic agents .
the experiment was conducted at the department of experimental surgery , federal university of pernambuco , using albino wistar male rats ( rattus norvegicus ) weighing 250  50  g , kept in individual cages of polypropylene measuring 30  20  19  cm and controlled lighting conditions ( 12  h light / dark photoperiod ) , temperature ( 24  2c ) , receiving water , and food ( labina ) ad libitum .
the experimental procedure was approved by the ethics committee on animal experimentation of the federal university of pernambuco ( case no . 23076.015015/2009 - 31 ) .
initially , 12 animals were weighed and intramuscularly preanesthetized with atropine sulfate ( 0.04  mg / kg ) and 10 minutes after subjected to anesthetic combination of 10% ketamine ( 90  mg / kg ) and 2% xylazine ( 10  mg / kg ) intramuscularly [ 15 , 16 ] . with the animal properly anesthetized trichotomy of back
thermal injuries were made with a solid aluminum bar 10  mm in diameter ( figure 1(a ) ) , previously heated in boiling water and so that the temperature reached 100c measured with a thermometer .
the bar is maintained in contact with the animal skin on the dorsal proximal region for 15  sec ( figure 1(b ) ) .
the pressure exerted on the animal skin corresponded to the mass of 51  g of aluminum bar used in the burn induction . immediately after the procedure
, analgesia with dipyrone sodium ( 40  mg / kg ) was performed intramuscularly , being maintained for three consecutive days sodium dipyrone at 200  mg / kg orally administered in the drinking water supplied to animals .
the clinical course of skin lesions by burns was evaluated for 28 consecutive days based on the following aspects : blistering , swelling , redness , crust , bleeding , secretion , granulation tissue , and scar tissue .
the wound retraction was evaluated using a caliper in 7 , 14 , 21 , and 28 days after burn induction .
% wound contraction on day x = [ ( area on day 0  open area on day x)/area on day 0 ]  100 . microbiological evaluation was carried out using  swabs  in the injury area at the moment of surgery and respective days of biopsies .
this sample was transferred to a petri dish of 20  150  mm containing nutrient agar medium in a laminar flow chamber .
after 24  h incubation , plates inoculated in triplicate for each sample were evaluated .
this routine evaluation was performed to evaluate the degree of contamination of injuries . at the preestablished times for biopsy ( 7 , 14 , 21 , and 28 days after burn induction ) , three animals randomly selected underwent anesthesia combination of 10% ketamine ( 90  mg / kg ) and 2% xylazine ( 10  mg / kg ) , intramuscularly [ 15 , 16 ] for tissue samples collection .
euthanasia was performed by excessive doses of sodium pentobarbital intraperitoneally ( 100  mg / kg ) .  
tissue samples were immediately fixed by immersion in 4% formaldehyde ( v / v ) prepared in pbs ( 0.01  m , ph 7.2 ) , followed by routine histological processing paraffin embedding , microtomy with 4  m cuts , and masson 's trichrome staining .
histological study was performed by comparative descriptive analysis of the experimental groups in binocular optical microscope ( zeiss - axiostar model ) where were evaluated the evolution of skin healing after thermal trauma .  
the histological analysis was performed by independent pathologist who was experienced in the examination of burn wound specimens , in the following ways : ( 1 ) inflammatory response , characterized by the presence of polymorphonuclear leukocytes ( pnm ) , ( 2 ) granular tissue , characterized by the presence of fibroblasts , myofibroblasts , and neovascularization , ( 3 ) fibrosis , characterized by the density of collagen fibers identified by the intensity of blue color observed under optical microscopy due to staining by masson 's trichrome .
a score was made for all parameters evaluated :  = absent , + = mild presence , + + = moderate presence , and + + + = strong presence .
all results were expressed as mean values for group  standard deviation and analyzed considering p < 0.05 as statistically significant .
this experimental model was established to standardize thermal burn injuries in order to obtain injuries with the same size and depth degree .
the choice of wistar rats due to these animals shows a great ease of handling , accommodation and resistance to surgical aggressions , and infectious processes , with low mortality [ 19 , 20 ] .
however , the choice of male rats is due to variations in hormonal cycles in females that could intervene in the process of tissue repair .
the result of clinical evaluation showed no signs of infection , secretion , bleeding , or death in both groups .
infected wounds heal more slowly , reepithelisation is more prolonged , and there is also the risk of systemic infection . shaving the back of the animals
was performed by manual traction of hair ( figure 2(a ) ) thus preventing secondary skin lesions that often occurs by the use of laminated devices .
the option to induce only one burning in the dorsal - proximal aimed at preventing the animal itself could reach the burn so that altering the outcome of the clinical evaluation of lesions .
the use of individual aluminum bar for each animal in the experimental group is important in reducing the interval between the induction of a burn and another within the same group , thus avoiding large variations in the assessment of healing time .
the size of lesions showed uniform average distribution of 10  1  mm in diameter ( figure 2(b ) ) .
similar studies by heredero and colleagues   and meyer and silva   revealed that it is not possible to perform a perfectly uniform burn in all experimental rats . according to vale
, the burn depth depends on the intensity of the thermal agent , generator or heat transmitter and time of contact with the tissue , which is the determinant of the aesthetic and functional result of the burn .
caused thermal burns by using 5  cm aluminum plate heated to 130c , which were pressed into the skin of the back for 5 seconds .
however , this method can generate lesions with different depths depending on the pressure during the procedure . in our study ,
the pressure was equivalent to the mass of the aluminum bar ( 51  g ) there being no interference by researcher , thus ensuring the reproducibility of thermal injuries .
the standardization of procedures , systematization , and organization of knowledge about the interrelationships of models is necessary to provide more reliable knowledge advance .
the most common method for obtaining second - degree thermal burns uses hot water as heat transfer agent .
induced second - degree thermal burns on the back of rats using submersion in hot water ( 90c ) for 6 seconds . in this experiment ,
10% body surface of the animal was injured producing lesions of variable size . according to orgaes
et al . , burns when reaching 26% to 30% of total body surface area of these mice cause mortality rates of 40% after three days , 52.5% after 7 days , 57.5% after 15 days , and 62.5% after 25 days .
results of this study revealed thermal burns white in color , painful , with no bubbles , mild edema until the 3rd day after injury ( figure 2(b ) ) .
similar definition is reported by [ 29 , 30 ] that describes the deep second - degree burns and injuries that have pale color with pain in lower intensity compared to superficial second - degree burn . in our evaluation variation of the degree of hyperemia in the first three days of experiment that changed from slight to absent
the formation of a thick and dry crust was observed from day 3 after burn induction .
signs of the scar tissue formation at the edge of the lesion were observed from day 14 ( figure 2(d ) ) .  
the burn healing occurs by second intention , which is a slow process with high risk of infection , producing scar retraction , which depending on the area of injury can cause extensive scarring and consequently high cost in treatment .
the contraction of skin lesions occurs centripetally fromthe injury edges being caused by the action of myofibroblasts present at the site . in turn
the percentage of lesion contraction at the end of the experiment was 66.67  1.66% .
values obtained in this study are similar to those published by zohdi and colleagues , who observed 72.75  1.8% of reduction in control rats treated with hydrogel without drug ( placebo ) at 28 days of study ( figure 3 ) .
according to mandelbaum and colleagues , the mechanism of tissue repair is the integration of dynamic cellular and molecular processes involving biochemical and physiological phenomena aiming at ensuring tissue restoration .
for this reason , only the clinical evaluation of a burn injury does not provide information on the evolution degree of tissue healing , being of fundamental importance of the histopathologic evaluation of these lesions .
the histopathological findings confirmed the acquisition of deep second - degree burns based on the observation of total autolysis of both the dermis and epidermis , without reaching the hypodermis .
these data are consistent with reports of several authors who characterize it as deep second - degree burn injuries that cause partial or total destruction of nerve endings , hair follicles , and sweat glands [ 25 , 35 , 36 ] .
thermal injury was observed on the 7th day and extensive inflammatory exudate featuring an intense inflammatory reaction .
describe in their study the occurrence in the control group , treated with saline solution , an acute inflammatory process on the 6th day of evaluation . by day 14
the inflammatory response was classified as moderate with presence of macrophages , progressing to discreet at day 21 . by day
28 signs of inflammatory response in the animals evaluated was not observed ( table 2 ) .
tissue still presented a complete destruction of the dermis and epidermis and maintenance of the hypodermis ( figure 4(a ) ) on the 7th day after lesion induction .
histopathology section of the burned skin of control animals on 5th day showed denuded epidermis , diffuse infiltration of plasma cells , lymphocytes , and polymorphs .
after 14 days the histopathological evaluation revealed moderate autolysis of the tissue , with discrete neovascularization and fibroblast proliferation , with loose collagen fibers , not modeled with mild fibrosis and crust absence ( figure 4(b ) ) .
yaman and colleagues   confirm the presence of crust formed by remnants of necrotic tissue and infiltration of mononuclear cells on the 4th day of experimentation in the control group .
the crust detachment was only observed by these authors on the 14th day of study . by day 21
we observed the absence of autolysis , discrete neovascularization and intense fibroblastic proliferation , with dense collagen , not modeled and moderate fibrosis ( figure 4(c ) ) . by the end of the experiment at 28 days , histological observations showed incomplete reepithelialization of the injured tissue with autolysis and absent neovascularization , showing moderate fibroblastic proliferation and fibrosis with the presence of modeled dense collagen fibers ( figure 4(d ) ) . wound healing
includes number of stages like clotting , inflammation , granulation , fibrosis , arrangement of collagen with spasm of wound , and epithelization .
the time required for complete healing of deep second - degree burns , without the application of specific therapeutic agents , can be three to six weeks or more , and these burns will leave a scar tissue that may hypertrophy and contract itself [ 29 , 30 ] .
in this new model of second - degree thermal burns , injuries are easy to create and easily reproducible . there are similarities with the human second - degree burns in clinical and pathologic aspects .
thus , the animal model presented in this study is applicable in evaluating the use of therapeutic agents in the healing evolution of deep second - degree burns .
 thermal lesions were produced in 12 male wistar rats , positioning a massive aluminum bar 10  mm in diameter ( 51  g ) , preheated to 99c  2c/10  min . 
 on the back of each animal for 15  sec . 
 after 7 , 14 , 21 , and 28 days , animals were euthanized . 
 the edema intensity was mild , with no bubble and formation of a thick and dry crust from the 3rd day . 
 the percentage of tissue shrinkage at 28 days was 66.67  1.66% . 
 there was no sign of infection , bleeding , or secretion . within 28 days 
 reepithelialization was incomplete , with fibroblastic proliferation and moderate fibrosis and presence of modeled dense collagen fibers . 
 it is concluded that the model established is applicable in obtaining deep second - degree thermal burns in order to evaluate the healing action of therapeutic agents of topical use . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion