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Thirty Wistar rats were operated after distribution in 5 groups with different times of ischemia. Thermograms were obtained by using a infrared camera. The surgical technique has been standardized for all groups: abdominal cavity opening by a 5cm length incision in the midline, abdominal wall plans section and cavity exposure, and exteriorization of the intestine. In group I (control), it was proceeded only laparotomy without superior mesenteric artery ligature. After first thermogram done, incision was closed with continuing suture. In each rat in groups II, III, IV and V, the superior mesenteric artery was located at its origin on abdominal aorta, dissected and occluded with a vascular microclamp, subjecting the intestine to ischemia in variable times.
Osvaldo MalafaiaI; Marcos Leal BrioschiII; Sonia Maria Schneider AokiIII; Fernando Gallego DiasIV; Bruno Schneider GugelminV; Massao Schneider AokiVI; Yuki Schneider AokiVII
IPhD, Full Professor of Surgery, Coordinator of Principles of Surgery Post-Graduation Program, FEPAR, Curitiba-PR, Brazil
IIPhD, President of Brazilian Society of Thermology, Curitiba-PR, Brazil
IIIMaster in Sciences, Principles of Surgery Post-Graduation Program, FEPAR, Curitiba-PR, Brazil
IVMaster in Thermal Engineer, Federal University of Paraná (UFPR), Curitiba-PR, Brazil
VBiomedical Engineer, Duke-University, Durham, NC, USA
VIGraduate Student, FEPAR, Curitiba-PR, Brazil
VIIGraduate Student, UFPR, Curitiba- PR, Brazil
PURPOSE: To study thermal variations obtained through infrared image in rats, and to evaluate the relationship between intestinal ischemic time and histopathological findings.
METHODS: Thirty Wistar rats were operated after distribution in 5 groups with different times of ischemia. Thermograms were obtained by using a infrared camera. The surgical technique has been standardized for all groups: abdominal cavity opening by a 5cm length incision in the midline, abdominal wall plans section and cavity exposure, and exteriorization of the intestine. In group I (control), it was proceeded only laparotomy without superior mesenteric artery ligature. After first thermogram done, incision was closed with continuing suture. In each rat in groups II, III, IV and V, the superior mesenteric artery was located at its origin on abdominal aorta, dissected and occluded with a vascular microclamp, subjecting the intestine to ischemia in variable times.
RESULTS: Rats submitted to a 30 minutes ischemia presented reactive hyperemia, thermal differential of 1.8°C and normal pathological examination. The 1 hour ischemia produced reactive hyperemia with ischemic areas, thermal differential of 1.0°C and injuries at villosities' tips. However, the 90 minutes ischemia had not shown reactive hyperemia with large ischemic areas, thermal differential of -1.0°C and injury in the upper third of the villosities. The 2 hours ischemia demonstrated a severe ischemia, thermal differential of -2.0°C and injury throughout the all villosities' extension.
CONCLUSION: It has been possible studying thermal variations through infrared image in rats, showing correlation between thermal response in thermograms, ischemic time and histopathological findings.
Key words: Spectrophotometry, Infrared. Tissue Survival. Ischemia. Wound Healing. Rats.
OBJETIVO: Estudar as variações térmicas obtidas por meio da imagem infravermelha em ratos, e avaliar sua correlação com o tempo de isquemia intestinal e os achados histopatológicos.
MÉTODOS: Trinta ratos Wistar foram operados após distribuição em cinco grupos com diferentes tempos de isquemia. Os termogramas foram obtidos utilizando-se uma câmera infravermelha. A técnica operatória foi padronizada para todos os grupos, abertura da cavidade abdominal por uma incisão na linha média de 5 cm de comprimento com secção de todos os planos da parede abdominal e exposição da cavidade e as alças intestinais exteriorizadas. No grupo I (controle) foi feito apenas laparotomia sem ligadura da artéria mesentérica superior. Após realizado o primeiro termograma, foi fechada a incisão com sutura contínua. Em cada rato dos grupos II, III, IV e V a artéria mesentérica superior foi localizada em sua origem na aorta abdominal, dissecada com e ocluída por um microclampe vascular submetendo o intestino à isquemia em tempos variáveis.
RESULTADOS: Os ratos submetidos à isquemia de 30 minutos apresentaram hiperemia reativa, diferencial térmico de 1,8°C e exame anatomopatológico normal. A isquemia de 1 hora produziu hiperemia reativa com áreas de isquemia, diferencial térmico de 1,0°C e lesões na ponta das vilosidades. Já a de 90 minutos não demonstrou hiperemia reativa com grandes áreas de isquemia, diferencial térmico de -1,0°C e lesão no terço superior das vilosidades. A isquemia de 2 horas mostrou isquemia grave, diferencial térmico de -2,0°C e lesão em toda a extensão das vilosidades.
CONCLUSÃO: Foi possível estudar as variações térmicas por meio da imagem infravermelha em ratos, que mostrou haver correlação entre a resposta térmica dos termogramas, o tempo de isquemia e achados histopatológicos.
Descritores: Espectrofotometria Infravermelho. Sobrevivência de Tecidos. Isquemia. Cicatrização de Feridas. Ratos.
The intestine's viability evaluation is one of the most difficult situations through which passes the surgeon who works in emergences services1. When faced with cases of mesenteric ischemia, especially in doubtful ones, where it is feared to underestimate an intestinal segment with low blood perfusion preserving it into the abdominal cavity, there is a risk of progression to sepsis and death. In addition, also not desirable, ischemia's superestimation and extensive resections can cause intestinal failure, requiring total parenteral nutrition or intestinal transplantation.
A few centimeters preservation of the small intestine can determine difference between the patients be able to absorb adequate oral diet or rely on permanent parenteral nutrition2. Total parenteral nutrition has high costs and not always is available in public hospitals, as well as being an able treatment for infectious and metabolic complications.
Considering that clinical criteria such as coloring, peristalsis, pulse's presence in marginal arcades and sectioned edges' bleeding may be failure in up to 60%3, several methods more objective, have been used to predict intestinal viability as: injection of radioactive microspheres using technetium 994,5; coloring intravascular injection6,7,8,9,10,11,12; ultrasound Doppler13; ultrasound laser Doppler14,15,16,17,18; intestinal electromyography19,20,21,22,23; and surface oximetry3,24,25,26,27. These methods have been experimentally applied as intestinal viability indicators, however none has achieved widespread clinical use.
Thus, infrared image has its use highlighted by technique modernization and nowadays has become important tool for the study of human body temperature. Infrared image is a method that uses special camera, targeted to operative field, which detects infrared radiation emitted by the body to be examined.
This technique was experimentally used by Moss et al.28, who studied, in dogs, infrared image to evaluate the viability of revascularizated intestine, previously submitted to 2 ½ hours and 8 hours ischemia29. The thermograms showed reactive hyperemia and elevation of 1 to 4°C in intestinal surface temperature when compared to adjacent normal intestine30. In the intestinal segment subjected to an 8 hours ischemia there was no reactive hyperemia.
However, despite the great technological development applied to medicine in recent years, especially in image examination and in life support advances, mortality rates in cases of acute mesenteric ischemia remain high, around 60 to 80%, according to several authors31,32,33,34,35,36 experience, needing yet researches about more accurate techniques on fast and efficient assessment of mesenteric ischemia.
The aim of this research was to study thermal variations, obtained through infrared image, in Wistar rats subjected to different intestinal ischemia times, and to evaluate thermograms and its correlation with ischemia time and histopathological findings.
This is an experimental study, with sample subject's double-blind random selection. The experimental protocol was approved by the Research Ethics' Committee of Faculdade Evangélica do Paraná, and it was adopted the ethical principles for animal testing of the Animal Experimentation Brazilian College31, using Veterinary Anatomical Nomina (1983).
It was used 30 male rats (Rattus norvegicus, Rodentio, Mammalia, Wistar line) aged between 2.5 and 3 months, and weighing between 270 and 300 g, accommodated in cages with five animals in controlled temperature (22°C), with light/darkness cycle of 12/12 hours, receiving proper feed for the species and water ad libitum, three days before operation until the end of the experiment.
They were anesthetized with ketamine hydrochloride at dose of 15 mg/kg by intramuscular, on the inner face of the pelvis member. Intraperitonial additional doses were administered as needed. After reflexes' loss, animals were weighed. The experiments were conducted in laboratory during the morning due to animal's lower thermal variation in that period. The study was conducted in heated room (22°C) controlled by air conditioner, with humidity of 60% controlled by air dehumidifiers and without current air. Heat loss by forced convection was minimized avoiding moving around the animals. Doors and windows were maintained closed but with gaps to air circulation towards stabilizing environment's temperature by air conditioner.
After anesthesia, the 30 rats were randomly distributed into 5 groups (Table 1). It was considered intestinal ischemia the superior mesenteric artery occlusion in its origin at abdominal aorta artery.
Initial surgical technique has been standardized for all groups with abdominal cavity opening by a 5 cm length incision in the midline, all abdominal wall plans section and cavity exposure. Intestine was exteriorized by the right protected by saline humid gaze and disguised with plastic film (Royal-pack®), reducing drying caused by contact with the external environment. In group I (control), it was done only laparotomy without superior mesenteric artery ligature. After first thermogram was done, the incision was closed with continuing suture and mice were revived from anesthesia. In each rat of groups II, III, IV and V, the superior mesenteric artery (Figure 1) was found in its origin at abdominal aorta, dissected and occluded by a 5 mm vascular microclamp, subjecting intestine to ischemia.
Operation had begun by the 6 animals in group V whose ischemia time was of 2 hours. Before arterial clamping, it was obtained intestine's thermograms of all animals. During pinching period, in their respective groups, intestine was replaced in abdominal cavity and incision was temporarily closed by single plan continuous suture to prevent liquid loss through evaporation. Rats were revived from anesthesia and kept in cage with identification. Same procedure was performed in other groups. After each group's previewed time (Table 1) abdomen was reopened, extruding up the intestine and microclamp was withdrawn, freeing the artery occlusion. It was waited up for 10 minutes, allowing thermal equilibrium with the environment, and thermograms were performed by using infrared image in each group of six animals to monitor small intestine's revascularization or not, up to 15 minutes.
The infrared camera was positioned up to 60 cm of surgical incision where small intestine was exposed to be evaluated. In this study, it was used an infrared camera (ThermaCamTM S65 - FLIR Systems TM - Sweden) to capture electromagnetic waves spectral band emitted between 3.5 to 5µ m, infrared waves. The maximum spatial resolution obtained was 0.1mm. Infrared radiation naturally emitted from the examined surface was converted, through a platinum silicate detector (PtSi) cooled with liquid nitrogen (steerling cicle), in electrical signal. This signal was processed in a 76,000 points spreadsheet number of calibrated absolute temperature, seven frames per second, instantly represented in thermal image with a 320x240 pixels resolution and thermal sensitivity up to 0.08°C. Camera was directly focused on the rat's ventral surface, forming an angle of 80° (Figure 2).
The images were processed in a 7500 MHz Pentium III computer attached to the PCMCIA card. Through specific program for image obtained analysis, the external surface irradiated temperatures were recorded at seven frames per second throughout the experiment. All images were represented by infrared thermograms in a video display and recorded in hard disk for later statistical analysis done by the program.
The images were analyzed by using temperature range between 35.0°C and 25.0°C, average temperature of 28.5°C and continuous colorimetric scale rain (Figure 3), in which the colors white, red, yellow, green, blue and black, represented respectively a decreasing scale of temperature areas, also distributed on the scale, from warmer to cooler, maintained until the experiment's ending.
After experiment's ending, rats were sacrificed for deepening of the anesthetic dose. The small intestine was removed and a 3 cm segment from the jejunum was collected, at about 10 cm from duodenojejunal angle, put into vials containing formalin (formaldehyde 10%) and encrypted for histological examination. Material received histological processing by conventional technique. The preparations were cut in a paraffin microtome, with 4 micrometers thick, and stained by the hematoxylin and eosin (HE) method.
Calculating formula for dimensionless values
The dimensionless thermal differential (ΔT) was obtained by following this formula43:
* Δ T is the dimensionless thermal differential;
* T is the experimental acquired temperature by infrared image;
* T ∞ is the temperature within the laboratory;
* T Ь is the average central temperature.
Calculating formula for cooling speed
* S cool is the cooling speed;
* Δ T is the dimensionless thermal differential;
* t is the time established in this study that was of 15 minutes.
For statistical analysis, it was used specific program STATISTICA, Version 6 (Tulsa, OK, Stat Soft, Inc). For thermal variation analysis, it was used average and uncertainty of measurement (T±UT), and for heat loss rate, average and twice the standard deviation (x±2sd). Data were tabulated, and gaussians attributes, independence and consistency of variables, were tested. When data met the proposed criteria, they were subjected to tests of univariate analysis ANOVA and Tukey's parametric, to establish the results significance. In all tests, it was set up the interval 0.05 or 5% (P < 0.05) to exclude the nullity hypothesis.