- Email: [email protected]
Quantification of burn induced extravasation of Evans blue albumin based on digital image analysis
PERGAMON
Computers in Biology and Medicine 28 (1998) 153±167
Quanti®cation of burn induced extravasation of Evans blue albumin based on digital image analysis Anders JoÈnsson a, Ulf Mattsson b, Jean Cassuto a, Guy Heyden b a
b
Department of Physiology, GoÈteborg University, GoÈteborg, Sweden Department of Oral Pathology, GoÈteborg University, GoÈteborg, Sweden Received 22 September 1995
Abstract The present study evaluated a non-invasive method based on digital image colour analysis that allows near-continuous quanti®cation of extravasated Evans blue albumin after burn injury. A full-thickness burn was induced in the abdominal skin of rats, followed by injection of Evans blue dye. Tissue content of Evans blue were quanti®ed using spectrophotometry, and compared with digital colour analysis. The non-invasive technique demonstrated a good correlation when compared to invasive spectrophotometry, but is superior by allowing near continuous measurement in the same subject and may be of value for evaluation of eective burn oedema-reducing treatment. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Burns; Oedema measurement; Computerised image analysis; Spectrophotometry; Evans blue; Rats
1. Introduction Burn injury results in a local in¯ammatory reaction involving the release of mediators, such as histamine [1±3], serotonin [4], kinins [5], prostaglandins [6±12], leukotriens [13, 14], complement factors [15±18] and neurogenic factors [19] resulting in vascular permeability changes and oedema formation. A variety of methods have been described to evaluate burn-induced plasma extravasation. Most of these methods are based on invasive techniques utilising colloid dyes and radioactive tracers [20±22], tissue biopsies [23] or wet/dry weight ratio [24]. All of these methods require that the experimental tissue is removed, consequently not allowing repetitive measurements in the same animal during the course of time. A non-invasive standardised method may therefore prove valuable for the study of the course of burn-induced tissue oedema and the evaluation of burn treatments aiming at reducing ¯uid losses. 0010-4825/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 1 0 - 4 8 2 5 ( 9 7 ) 0 0 0 3 8 - 3
154
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
Computerised image analysis is currently being used in several ®elds of medical research and examinations. The use of photographic analysis for non-invasive diagnosis of burn depth is described as early as 1977 [25]. A technique of using image analysis for measurements of erythema and blood ¯ow has recently been described by Mattsson et al. [26]. Computerised image analysis has also been employed in clinical follow-up of burn patients [27] and for ¯uorescence digital microscopic evaluation of macromolecular diusion in burn injury [28, 29]. The aims of the present study were to elaborate and evaluate a non-invasive method that allowed continuous or near continuous, objective measurements of extravasated plasma albumin marked with Evans blue in a tissue exposed to a standardised burn injury, and to establish whether the obtained values from image feature analysis correlate with invasive measurements from spectrophotometric analysis of extravasated Evans blue albumin.
2. Materials and methods 2.1. Animals and materials Experiments were performed on 38 male Sprague-Dawley rats weighing 330±340 g. The protocols were approved by the regional Animal Care Committee. Animals were housed for at least 7 days prior to the experiments in a ventilated and temperature-controlled room and had water available ad libitum. The day±night cycle was constant at 12 h light and 12 h dark. Anaesthesia was induced with pentobarbital (50 mg/kg) intraperitoneally and maintained by a continuous intravenous infusion of chloralose (1.5 mg kgÿ1 minÿ1). A tracheostomy was performed and a tracheal cannula was inserted to secure free airway. Blood pressure was monitored using a pressure transducer (Statham P23 AC) connected to a cannula in a femoral artery. Heart rate was measured using a heart rate meter. Body temperature was kept at 388C by placing the animal on a thermoregulated heating pad. 2.2. Thermal trauma model Full-thickness, second degree burn injury was induced in the closely shaved abdominal skin by a technique previously described by Cassuto et al. [22] using an electrically heated aluminium rod with a bottom surface of 1 1 cm connected to an adjustable transformer. A thermosensor electrode from the aluminium rod was connected to a chartwriter (Metrawatt SE 120, ABB, Switzerland) for temperature registration. In order to calibrate the system, room temperature was set on the chartwriter against a thermometer. Room temperature was in the range of 21±238C and was used as the ®rst ®x point on the calibration curve. The bottom surface of the thermoprobe was subsequently sunk into water (5 ml) and heated by turning on the current until the water boiled. At this point the calibration curve levelled o and the level for 1008C was set on the chartwriter, serving as the second ®x point on the calibration curve. Current was cut o and the hot plate was removed from the water, dried, and allowed to cool in the air. When the temperature reached 558C the probe was put in contact with the abdominal skin until the temperature reached 458C, at which time the probe was removed. This procedure allowed a constant amount of heat to be administered to each animal
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
155
independent of possible variations in skin temperature. Probe pressure was standardised to 1.2 N/m2 by pushing down a cylindrical handle compressing in turn a spring on the probe. Evans blue dye (20 mg/kg dissolved in 1 ml of isotonic saline) was administered intravenously to 30 animals 60 min after the burn. At the end of the experiments (180 min) the animals were killed by an intravenous injection of 1 ml saturated KCl. The thermally injured skin area (1 1 cm full thickness burn injury) of Evans blue-treated rats was dissected fullskin, dried on ®lter paper to remove excess ¯uid, and immediately weighed. Each sample was placed in 4 ml formamide (HCONH2) and incubated for 24 h in a water bath at 508C as described by Gamse et al. [30]. Colorimetric measurements were performed on the ¯uid in a Stasar1 (Gilford, U.S.A.) spectrophotometer at the peak absorption of 612 nm. Three measurements were made on each sample and the mean value was used for calculations based on external standards in formamide. 2.3. Fluorescence microscopy preparation The experimental area of four burned animals and the corresponding skin area of four nonburned control animals were prepared for ¯uorescence microscopy as described previously [31]. Evans blue dye was injected intravenously 60 min after the thermal trauma. Two hours later the experimental area was dissected fullskin and the specimens were ®xed in 4% buered formalin for 24 h. Following this procedure the tissue specimens were frozen in liquid nitrogen, sectioned into 10 mm slices in a cryostat (Kryostat 1720, Leitz), mounted on glass slides and dried by heating to 308C for 1 min. The sections were examined in a ¯uorescence microscope (Microphot-FX, Nikon, lens Fluor 10 , ®lter ND 4) using an epi-illumination technique (Ploek Pam System) consisting of a UV-lamp, an excitation ®lter (450±490 nm), a beamsplitting mirror (510 nm) and a suppression ®lter (515 nm). Photographs were taken using ASA 160 ®lm (Kodak Ektachrome 135-36). All specimen analysis was performed by an assistant blinded to the experimental protocol. 2.4. Photographic technique and equipment The technical equipment used for the photographic documentation consisted of a 35 mm single lens re¯ex camera (Contax 167 MT) with state-of-the-art electronic apparatus, except for the automatic focusing device. The camera was provided with a data back for the printing of the exact time of photography on each colour slide. Using a 100 mm macro lens (Carl Zeiss Makro-Planar T, f 2.8) and a telephoto converter (Carl Zeiss Mutar T1 2) a 200 mm optical system with comfortable working distance was achieved. A double electronic ¯ash unit (Combi¯ash, guide number 50 at 100 ISO) was used. Through-The-Lens (TTL) electronic system of the camera limited the exposure deviations. An area of the skin adjacent to and at least 20 mm from the burn area was included in all photographs. Two photographs were taken at each interval with 1 min between in order to evaluate possible exposure deviations. The technical devices and procedures for standardised clinical macrophotography have been described in detail by Eliasson and Heyden [32]. Photographs of
156
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
the shaved abdominal skin were taken immediately before inducing the burn injury (referred as 0 min) and then at 5, 60, 65, 90, 120, 150 and 180 min after the burn trauma. A 36 exposure slide ®lm (Agfachrome 200 RS Professional) was used for the photographs. The ®lms were developed and mounted in plastic frames at a standard ®lm laboratory (Agfachrome Service, Herrljunga, Sweden). Prior to inducing the burn injury, the area where the thermal probe was to be put in contact with the skin was identi®ed and marked with ink. This was done in order to facilitate the identi®cation of the area where image features were to be extracted in the digitised images.
2.5. Image digitisation The colour slides were digitised using an image scanner (Dixel 2000, Hasselblad Electronic Imaging AB, GoÈteborg, Sweden). The digitised images had a resolution of 512 368 pixels with 24 bits per pixel and 8 bits per colour separation. Each pixel had Red (R), Green (G) and Blue (B) values on a scale from 0 to 255. The digital information was then transmitted to an IBM compatible PC (Victor 486 DX) with an extended graphics card (AT Vista Truevision Inc., Indianapolis, U.S.A.) for image feature extraction and analysis. The software designed for this purpose was developed in collaboration with Hasselblad Electronic Imaging AB (GoÈteborg, Sweden).
2.6. Image feature extraction The border of the burn area was identi®ed and outlined and an area of approximately 10 000 pixels was created (Fig. 1). The location and co-ordinates of each created object in each slide was stored on the hard disc of the computer. The same object was then used and positioned in the corresponding burn area on subsequent slides taken.
Fig. 1. Clinical appearance of rat skin prior to thermal injury (left) and 180 min postburn (right), i.e. 120 min after IV administration of Evans blue dye. The areas where image features were extracted in the digitised images are framed.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
157
2.7. Colour systems Two dierent colour systems were used in the image feature analysis. The purpose was to evaluate whether any of the systems presented advantages for the biological interpretation of the observed colour dierences, but also to establish whether correlation existed between two dierent colour features and data obtained by spectrophotometric analysis of extravasated Evans blue albumin. The colour systems used were:
2.7.1. Normalised red (n-r), green (n-g) and blue (n-b) values (rgb) The colour system describes the relative amount of Red (R), Green (G), and Blue (B) respectively for each pixel. R 0Rn ÿ rR255 n ÿ r 255 RGB
Fig. 2. Appearance of rat skin prior to thermal injury (upper diagrams) and at 120 min after administration of Evans blue dye (lower diagrams). The distribution of mean values for normalised-red, -green, -blue and Intensity, Hue and Saturation values from left to right in the marked areas are demonstrated. Normalised rgb (left): Note the increase in normalised-blue value and the decrease in normalised-red value in the burned area with extravasated Evans blue. Intensity-Hue-Saturation (right): The blue discoloration in the thermally injured area is demonstrated by an increase of Saturation value and a decrease of Hue and Intensity values when compared to values prior to thermal injury.
158
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
n ÿ g 255
G RGB
n ÿ b 255
B RGB
0Rn ÿ gR255 0Rn ÿ bR255
For example, an area with a high normalised blue (n-b) value and low normalised red (n-r) and green (n-g) values is visually perceived as a blue colour (Fig. 2, lower left diagram).
2.7.2. Hue(H)-Saturation(S)-Intensity(I) values (HSI) The colour system is built on perceptive parameters. The distribution of colours is shown in Fig. 3. The hue parameter describes the colour, for example, red or blue. The colours are arranged in a radial pattern and each hue can consequently have a value ranging from 0 to 360 degrees. Intensity is a colour-neutral parameter that describes the relative lightness or darkness of the hue. Saturation shows how much the hue is mixed with white. A highly saturated colour contains very little white and the saturation consequently increases towards the periphery of the circle. The values for Intensity-Hue-Saturation were calculated as follows (modi®ed from Ledley [33]). Hue p GÿB 180 0RH<360 H Arc tan 3 2R ÿ G ÿ B Saturation S 1 ÿ 3 minr,g,b 255 0RSR255 [r,g,b] means the value for one of n-r, n-g or n-b.
Fig. 3. Distribution of Hue values in the HSI-colour system. Note the location of the red and blue colours. The Saturation increases towards the periphery of the circle.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
159
Intensity I
RGB 0RIR255 3
Mean values and standard deviations for the colour features described above were measured within each created object and stored in a separate ®le for subsequent statistical analysis. Tissue colour changes were expressed as the dierence in mean values for the colour parameters from one interval to the next. The changes in HSI values due to colour changes are shown in Fig. 2 (right diagrams). 2.8. Comparison of the two colour systems A comparative analysis of the two colour systems was performed in order to establish whether linear correlation existed between the changes observed for the image parameters in order to interpret the clinical impression of colour changes. 2.9. Statistical analysis Paired two-tailed Student's t-test was used in the statistical analysis and a p-value less than 0.05 was regarded as statistically signi®cant. The comparison between image features and spectrophotometric values was performed by linear correlation analysis.
3. Results 3.1. General One animal died during the ®rst hour of the experiments. Two tissue specimens subject to spectrophotometric analysis were lost due to identi®cation problems, leaving 27 samples for the correlation analysis. No cardiovascular eects of the burn trauma or the administration of Evans blue were observed during the experiments. 3.2. Fluorescence histology Skin specimens from four burned animals subject to ¯uorescence histology demonstrated rich Evans blue ¯uorescence in all layers of the skin, whereas in four non-burned control animals the ¯uorescence was mainly localised in the lumen and walls of blood vessels. 3.3. Spectrophotometry Administration of Evans blue 60 min after the thermal trauma resulted in a signi®cant blue coloration of the skin at 180 min postburn. The amount of Evans blue in the skin was 59.129.3 ng/mg tissue (mean2SEM, n = 27).
160
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
3.4. Normalised rgb values The colour changes during the course of the experiment are shown in Fig. 4. During the interval 0±5 min only minor and statistically not signi®cant changes were recorded for the 29 animals. From 5 to 60 min, small but statistically signi®cant changes were observed. Changes comprised of a small decrease in n-r value and a minor increase in n-b value. At 65 min postburn, i.e. immediately after intravenous administration of Evans blue the colour pattern showed fast increases in n-g and n-b values and a corresponding decrease in n-r value. These changes were statistically signi®cant ( p < 0.001). The major colour dierences were, however, observed in the intervals between 65±90 min and 90±120 min. During these intervals there was a continuous increase in n-b value and decrease in n-r value ( p < 0.001). Between 120 and 150 min the visible changes were smaller but statistically signi®cant for n-r and n-b values ( p < 0.05). In the last interval from 150 to 180 min, the observed changes were not signi®cant. For the group as a whole, the n-g value remained relatively constant during the entire period of observation. 3.5. Hue-Saturation-Intensity As shown in Fig. 5, only minor changes in HSI values were found in the interval from 0 to 5 min. During 5±60 min minor but statistically signi®cant ( p < 0.001) decreases in Hue and Saturation values were detected. From 60 to 65 min, a further decrease in Hue value was observed together with a minor drop in Saturation ( p < 0.001). As mentioned above, the most prominent colour changes in the burn area were observed in the intervals from 65 to 90 min and 90 to 120 min ( p < 0.001). During these periods a continuous decrease in Intensity and Hue values were detected, paralleled by an increase in Saturation values. The changes were of
Fig. 4. The distribution of mean (2SEM) pixel values for normalised-red, -green and -blue (n-r, n-g, n-b) in thermally injured rat skin. Note the changes for n-b values and for n-r values after administration of Evans blue dye at 60 min post burn.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
161
Fig. 5. Changes in the Intensity-Hue-Saturation colour system (mean 2 SEM) after thermal injury to rat skin. Note the changes in all three parameters occurring after administration of Evans blue dye at 60 min post burn.
comparable magnitude in the two intervals. After 120 min only minor and statistically not signi®cant changes could be detected. 3.6. Comparison of the two colour systems Correlation analysis showed that there was a high linear correlation between the changes in normalised rgb values respectively and dierences in Intensity-Hue-Saturation values from 0 to 180 min (Table 1). Decreases in Intensity and Hue values correlated signi®cantly with decreases in n-r and n-g values and an increase in n-b value. An increase in Saturation was correlated to decreased n-r and n-g values and increased n-b value. 3.7. Comparison between spectrophotometry and digital image analysis Correlation analysis of Evans blue spectrophotometry values and of changes in n-b values 60±180 min postburn, showed a high linear correlation coecient of 0.89 ( p < 0.001) (Table 2). 3.8. Methodological errors Image feature values from two consecutive photographs of a corresponding area were compared. The analysis revealed that the dierences between exposures were not signi®cant and had no in¯uence on the interpretation of the results (Table 3). Table 1 Correlation analysis between two colour systems. Results from linear correlation analysis between measured dierences in image feature values from 0 to 180 min for normalised-red, -green and -blue values (n-r, n-g, n-b) and Hue-Saturation-Intensity (HSI) values
Hue Saturation Intensity
n-r
n-g
n-b
0.83 *** ÿ0.77 *** 0.55**
0.37 * ÿ0.71 *** 0.83 ***
ÿ0.80 *** 0.87 *** ÿ0.73 ***
* = p < 0.05, ** = p < 0.01, *** = p < 0.001, n = 27.
162
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
Table 2 Correlation analysis between spectrophotometry and the DIA technique. Correlation coecients and probability values after linear correlation analysis between spectrophotometry values and normalized-red, -green and -blue values (n-r, n-g, n-b) and Intensity-Hue-Saturation values, respectively
Spectrophotometry Spectrophotometry
n-r
n-g
n-b
ÿ0.82 *** Hue ÿ0.59 **
ÿ0.64 *** Saturation 0.85 ***
0.89 *** Intensity ÿ0.69 ***
** = p < 0.01, *** = p < 0.001, n = 27. Table 3 Methodological error values of the DIA technique. Mean dierences in normalised-red, -green and -blue values (n-r, n-g, n-b) and Hue-Saturation-Intensity (HSI) values measured in corresponding areas in two consecutive photographs at the same time interval (n = 29). Mean dierences were not signi®cant Normalised values
n-r
Mean dierence SD SEM
0.20 1.24 0.21
HSI-values Mean dierence SD SEM
Hue 0.12 0.77 0.13
n-g 0.04 0.39 0.07 Saturation 0.71 3.64 0.63
n-b 0.24 1.21 0.21 Intensity 0.30 4.78 0.83
4. Discussion Skin burn injury involves a wide range of vascular changes often leading to increased permeability and subsequent oedema formation [4]. Traditionally, experimental animal models investigating burn-induced oedema have used invasive methods such as tissue wet/dry weight [24, 34], and analysis of Evans blue albumin extravasation by spectrophotometry [22, 35]. A few non-invasive methods, such as dichromatic absorptiometry [23], estimation of lymph ¯ow from burned areas [11, 36, 37] and measurement of rat limb volume [38] have also been described. Invasive methods are generally accepted to be more exact but require that tissue specimens are taken from the animals thereby not allowing continuous evaluation of progressive changes in the burn area of the same animal. Moreover, invasive methods cannot be employed in human subjects, necessitating the development of sensitive non-invasive methods for the study of burn pathophysiology and evaluation of burn treatments. In the present study a non-invasive technique was developed allowing repetitive evaluation of colour changes in an area of thermal injury. Our objective was to decide whether such colour changes were representative of the pathophysiology of experimental burn trauma and if quanti®cation and statistical analysis of physiological and pathophysiological processes could be accurately performed. Results showed that intravenous administration of Evans blue dye resulted in a pronounced blue discoloration of the experimental area (Fig. 1) suggesting a pronounced accumulation of
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
163
extravasated Evans blue albumin. The distribution of Evans blue albumin in the burned area was analyzed by ¯uorescence microscopy and revealed that large amounts of Evans blue had accumulated in all layers of the skin supporting the fact that this is a full-thickness burn. In non-burned control animals, however, Evans blue was mainly located inside the blood vessels, suggesting that the bluish discoloration of the surrounding non-burned skin was representative of intravasal non-extravasated Evans blue albumin. Normal, shaved rat skin contains almost equal amounts of red, green and blue colour, with a slight domination of red (Fig. 2, upper left panel). Repeated photographs of the burned area followed by normalised rgb analysis allowed a near continuous monitoring of EB-albumin extravasation in the burned tissue. Results showed a continuous increase in n-b values and a corresponding decrease in n-r, while n-g remained virtually unchanged with time (Fig. 4). Since normalised rgb values have been used, an isolated increase in the blue colour must result in an equivalent decrease of the sum of the other two colours. These results were in line with colour changes observed by the naked eye showing continuously increased blue coloration of the burned skin. Correlation analysis between values obtained by spectrophotometric quanti®cation of Evans blue in the burned skin and normalised rgb values obtained by the DIA technique showed signi®cant correlation for all tested variables (Table 2). A negative correlation was seen for n-r and n-g due to the decreasing values for these variables as opposed to increasing spectrophotometric values due to increased EB-leakage. Despite small changes in the normalised green values, a signi®cant negative correlation was obtained due to the high sensitivity of the DIA technique. Since both n-b and spectrophotometric values increased towards the end of the experiment, a positive correlation was found (Table 2). The dierence in n-b values between the time of administration of Evans blue (60 min postburn) and the end of the experiment (180 min postburn) can thus be used as a quantitative expression of oedema formation in the same way as spectrophotometry analysis of accumulated extravasated plasma albumin in the experimental skin area. However, since spectrophotometric analysis in this study is performed on the full-skin preparation, data are representative of the total content of Evans blue albumin in the burned skin tissue. The question is whether colour changes observed by the DIA technique are representative of changes in all skin layers or only in the skin surface. Rat skin is rather translucent, since subcutaneously located blood vessels can be observed by the naked eye, suggesting that the colour changes monitored by the DIA technique are probably representative of colour changes in the full thickness of the skin. The same colour changes could also be expressed using the HSI system (Fig. 5). Evans blue accumulation in the burned tissue resulted in decreased Intensity values in the burned area corresponding with an observed darkening of the experimental area (Fig. 5). In normal rat skin, the Hue value lies near 1808, i.e. close to the red colour (Fig. 3). In connection with increased blue coloration of the burned area the Hue value decreased towards 608 and, as expected, ending in the blue sector. Saturation is inversely related to the amount of white colour. In normal rat skin the Saturation values are low since the skin appears whitish. This relationship leads to increased Saturation values due to decreased amount of white following accumulation of blue colour (Fig. 5). The blue discoloration could be expressed as a continuous decrease in Intensity and Hue values concomitant with an increase in Saturation values. All of these changes signi®cantly correlated with spectrophotometric values (Table 2).
164
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
These data suggest that Saturation values and the inversed values of Intensity or Hue can independently be used as quantitative variables describing the continuous accumulation of Evans blue albumin in the burned skin. The methodological errors associated with the DIA technique were limited and did not have any signi®cant in¯uence on the results when expressed as the standard deviation (SD) between the values obtained in two consecutive exposures (Table 3). The camera equipment used for photographic registration combined with an image scanner which converted the slides into digital images demonstrated a reproducibility which was adequate and sucient for the experimental purposes of the present study, as suggested by low mean dierences (less than 5%; Table 3). Since the DIA technique is based upon analysis of visible colour in the skin, it is essential to shave as close to the skin as possible in order to reduce the disturbing eects of shading. It must be stressed that during preparation of the animals care must be taken not to mechanically traumatise the skin since it would jeopardise the possibility of performing adequate measurements with the digital technique. In conclusion, both normalised rgb values and HSI values showed signi®cant correlation with spectrophotometric values (Table 2). These results suggest that the non-invasive DIA technique can be used as an alternative to invasive spectrophotometric analysis in quantifying Evans blue albumin accumulation in burned tissue. The DIA technique is, however, superior to invasive methods by allowing repeated measurements in the same area without interfering with the pathophysiological processes. In addition, correlation analysis between the two colour systems showed a signi®cant correlation between normalised rgb values and HSI values, suggesting that both systems can be used independently for repetitive monitoring of tissue colour changes.
5. Summary Profuse oedema formation is one of the main characteristics of skin burn. Most methods used to quantify experimental burn oedema are based on invasive techniques which do not allow repeated measurements in the same tissue. The present study evaluated a non-invasive method based on digital image analysis that allows near-continuous quanti®cation of extravasated Evans blue albumin in a standardised burn model. A standardised 1 cm2 full-thickness 2nd degree burn injury was induced in the abdominal skin of 30 anaesthetised rats. One hour post-burn the animals received a single injection of Evans blue (EB) dye. Two hours after the EB-injection, the burn area was dissected full skin and its contents of Evans blue were quanti®ed using spectrophotometry. Colour slides of the burn area were digitised and two dierent colour-systems (normalised-rgb values and the HueSaturation-Intensity system) were used to quantify skin contents of Evans blue and correlated to spectrophotometric data. Correlation coecients for spectrophotometry versus normalised blue were 0.89 ( p < 0.001), normalised red ÿ0.82 ( p < 0.001) and normalised green were ÿ0.64 ( p < 0.001), respectively. Correlation coecients for Hue-Saturation-Intensity system vs spectrophotometry were: Hue ÿ0.59 ( p < 0.01), Saturation 0.85 ( p < 0.001), Intensity ÿ0.69 ( p < 0.001). Methodological errors were negligible.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
165
We have developed a non-invasive method for quantifying experimental burn oedema. The present non-invasive technique for quantifying experimental burn oedema demonstrated a good correlation when compared to invasive spectrophotometry, but is superior by allowing near continuous measurement in the same subject and may be of value for evaluation of eective burn oedema reducing treatment.
Acknowledgements This work was supported by grants from the Bohus County Council and the Medical Society of GoÈteborg.
References [1] J.V. Boykin Jr.., E. Eriksson, M.M. Sholley, R.N. Pittman, Histamine-mediated delayed permeability response after scald burn inhibited by cimetidine or cold-water treatment, Science 209 (1980) 815. [2] J.V. Boykin Jr.., S.L. Crute, Inhibition of increased serum histamine and lactate after severe scald injury and cold-water treatment, Curr. Surg. 38 (1981) 393. [3] J.V. Boykin Jr.., E. Eriksson, M.M. Sholley, R.N. Pittman, Cold-water treatment of scald injury and inhibition of histamine-mediated burn edema, J. Surg. Res. 31 (1981) 111. [4] W.G. Cio Jr.., J.E. DeMeules, R.L. Gamelli, Vascular reactivity following thermal injury, Circ. Shock 25 (1988) 309. [5] M.S. Starr, G.B. West, Bradykinin and oedema formation in heated paws of rats, Br. J. Pharmacol. 31 (1967) 178. [6] G. Arturson, M. Hamberg, C.-E. Jonsson, Prostaglandins in human burn blister ¯uid, Acta Physiol. Scand. 87 (1973) 270. [7] D.N. Herndon, S. Abston, M.D. Stein, Increased thromboxane B2 levels in the plasma of burned and septic burned patients, Surg. Gynecol. Obstet. 159 (1984) 210. [8] R.H. Demling, C. LaLonde, Topical ibuprofen decreases early postburn edema, Surgery 102 (1987) 857. [9] T.J. Williams, Prostaglandin E2, prostaglandin I2 and the vascular changes of in¯ammation, Br. J. Pharmacol. 65 (1979) 517. [10] J.P. Heggers, F. Ko, M.C. Robson, R. Heggers, K.E. Craft, Evaluation of burn blister ¯uid, Plast. Reconstr. Surg. 65 (1980) 798. [11] C.-E. Jonsson, Y. Shimizu, B.B. Fredholm, E. GranstroÈm, E. Oliw, Eux of cyclic AMP, prostaglandin E2 and F2a and thromboxane B2 in leg lymph of rabbits after scalding injury, Acta Physiol. Scand. 107 (1979) 377. [12] J.P. Heggers, G.L. Loy, M.C. Robson, E.J. Del Beccaro, Histological demonstrations of prostaglandins and thromboxanes in burned tissues, J. Surg. Res. 28 (1980) 110. [13] C. Denzlinger, S. Rapp, W. Hagmann, D. Keppler, Leukotrienes as mediators in tissue trauma, Science 230 (1985) 330. [14] F. Alexander, M. Mathieson, K.H.T. Teoh et al, Arachidonic acid metabolites mediate early burn edema, J. Trauma 24 (1984) 709. [15] W.J. Schirmer, J.M. Schirmer, G.B. Na, D.E. Fry, Complement-mediated hemodynamic depression in the early postburn period, J. Trauma 29 (1989) 932. [16] K.T. Oldham, K.S. Guice, G.O. Till, P.A. Ward, Activation of complement by hydroxyl radical in thermal injury, Surgery 104 (1988) 272. [17] K.T. Oldham, K.S. Guice, G.O. Till, P.A. Ward, Evidence of local complement activation in cutaneous thermal injury in rats, Prog. Clin. Biol. Res. 264 (1988) 421.
166
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
[18] P.A. Ward, G.O. Till, Pathophysiologic events related to thermal injury of skin, J. Trauma 30 (Suppl.) (1990) S75. [19] E.T. Wei, J.G. Kiang, Peptides of the corticoliberin superfamily attenuate thermal and neurogenic in¯ammation in rat pawskin, Eur. J. Pharmacol. 168 (1989) 81. [20] W.W.L. Glenn, J. Muus, C.K. Drinker, Observations on the physiology and biochemistry of quantitative burns, J. Clin. Invest. 22 (1943) 451. [21] O. Cope, F.D. Moore, A study of capillary permeability in experimental burns and burn shock using radioactive dyes in blood and lymph, J. Clin. Invest. 23 (1944) 241. [22] J. Cassuto, P. NellgaÊrd, L. Stage, A. JoÈnsson, Amide local anesthetics reduce albumin extravasation in burn injuries, Anesthesiology 72 (1990) 302. [23] R.H. Demling, R.B. Mazess, R.M. Witt, W.H. Wolberg, The study of burn edema using dichromatic absorptiometry, J. Trauma 18 (1978) 124. [24] H. Onarheim, T. Lund, R. Reed, Thermal skin injury: II. Eects on edema formation and albumin extravasation of ¯uid resuscitation with lactated Ringer's, plasma, and hypertonic saline (2400 mosmol/l) in the rat, Circ. Shock 27 (1989) 25. [25] V.J. Anselmo, B.E. Zawacki, Multispectral photographic analysis. A new quantitative tool to assist in the early diagnosis of thermal burn depth, Ann. Biomed. Eng. 5 (1977) 179. [26] U. Mattsson, A. JoÈnsson, M. Jontell, J. Cassuto, Digital image analysis (DIA) of colour changes in human skin exposed to standardized thermal injury and comparison with laser doppler measurements, Comput. Methods. Programs Biomed. 50 (1996) 31. [27] D. Franceschi, R.L. Gerding, R.B. Fratianne, Microcomputer image processing for burn patients, J. Burn Care Rehabil. 10 (1989) 546. [28] S.J. Aggarwal, S.J. Shah, K.R. Diller, C.R. Baxter, Fluorescence digital microscopy of interstitial macromolecular diusion in burn injury, Comput. Biol. Med. 19 (1989) 245. [29] S.J. Aggarwal, K.R. Diller, H.K. Yeung, C.R. Baxter, Local macromolecular extravasation in thermal burns quanti®ed by ¯uorescent video microscopy and computer vision, J. Burn Care Rehabil. 15 (1994) 104. [30] R. Gamse, P. Holzer, F. Lembeck, Decrease of substance P in primary aerent neurones and impairment of neurogenic plasma extravasation by capsaicin, Br. J. Pharmacol. 68 (1980) 207. [31] A. Saria, J.M. Lundberg, Evans blue ¯uoroscence: quantitative and morphological evaluation of vascular permeablility in animal tissues, J. Neurosci. Methods 8 (1983) 41. [32] L. Eliasson, G. Heyden, Clinical macrophotography for oral health monitoring purposes. Methods and results from a comparative study, Swed. Dent. J. 14 (1990) 1. [33] Ledley, R.S., Buas, M., Golab, T.J., 1990. Fundamentals of true-color image processing. In: IEEE (Ed.), 10th International Conference on Pattern Recognition, Atlantic City, U.S.A., vol. 1, p. 791. [34] I. Blomgren, U. Bagge, Postburn blood ¯ow, edema, and survival of the hairy mouse ear after scald injury at dierent temperatures, Scand. J. Plast. Reconstr. Surg. 18 (1984) 269. [35] A. Saria, Substance P in sensory nerve ®bres contributes to the development of oedema in the rat hind paw after thermal injury, Br. J. Pharmacol. 82 (1984) 217. [36] G. Arturson, C.-E. Jonsson, Eects of indomethacin on the transcapillary leakage of macromolecules and the eux of prostaglandins in the paw lymph following experimental scalding injury, Ups. J. Med. Sci. 78 (1973) 181. [37] J.R. Casley-Smith, J.R. Casley-Smith, The eects of diosmin (a benzo-pyrone) upon some high-protein oedemas: lung contusion, and burn lymfoedema of rat legs, Agents Actions 17 (1985) 14. [38] G. Arturson, O.P. Jakobsson, Oedema measurements in a standard burn model, Burns Incl. Therm. Inj. 12 (1985) 1.
Anders JoÈnsson MD PhD is sta specialist at the Department of Orthopedic Surgery, Sahlgrenska University Hospital, MoÈlndal, Sweden and research fellow at the Department of Physiology, GoÈteborg University, GoÈteborg, Sweden. His research interest is the pathophysiology of trauma response and especially the response to thermal trauma.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
167
Ulf Mattsson DDS PhD works at the Clinic of Oral and Maxillofacial Surgery and Hospital Dental Care, Central Hospital, Karlstad, Sweden. His research has been focused on elaborating non-invasive methods for evaluation of skin and oral mucosal disorders. He is currently involved in research in the ®eld of Oral Medicine with special emphasis on pathogenesis and treatment of oral mucosal lesions. Jean Cassuto MD PhD is Research Director at Sahlgrenska University Hospital, MoÈlndal. He is also leading a research group at the hospital on several research areas including burns, pain, and in¯ammatory mechanisms. In addition to clinical research, the group is conducting experimental research at the Department of Physiology, GoÈteborg University. Dr Cassuto has been the tutor of eleven thesis-projects, eight of which have been presented at GoÈteborg University, Sweden. Guy Heyden OD MSc is Professor in Oral Pathology, UNESCO-Cousteau Ecotechnie Chairholder and Dean, Faculty of Thematic Studies, GoÈteborg University, Sweden. As Supervisor of the interdisciplinary Koster Health Project, Guy Heyden has developed standardized photographic methods for the monitoring of human health changes and neighbouring environmental variationsÐroutines facilitating retrospective studies utilizing, for example, digital image analysis.
Computers in Biology and Medicine 28 (1998) 153±167
Quanti®cation of burn induced extravasation of Evans blue albumin based on digital image analysis Anders JoÈnsson a, Ulf Mattsson b, Jean Cassuto a, Guy Heyden b a
b
Department of Physiology, GoÈteborg University, GoÈteborg, Sweden Department of Oral Pathology, GoÈteborg University, GoÈteborg, Sweden Received 22 September 1995
Abstract The present study evaluated a non-invasive method based on digital image colour analysis that allows near-continuous quanti®cation of extravasated Evans blue albumin after burn injury. A full-thickness burn was induced in the abdominal skin of rats, followed by injection of Evans blue dye. Tissue content of Evans blue were quanti®ed using spectrophotometry, and compared with digital colour analysis. The non-invasive technique demonstrated a good correlation when compared to invasive spectrophotometry, but is superior by allowing near continuous measurement in the same subject and may be of value for evaluation of eective burn oedema-reducing treatment. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Burns; Oedema measurement; Computerised image analysis; Spectrophotometry; Evans blue; Rats
1. Introduction Burn injury results in a local in¯ammatory reaction involving the release of mediators, such as histamine [1±3], serotonin [4], kinins [5], prostaglandins [6±12], leukotriens [13, 14], complement factors [15±18] and neurogenic factors [19] resulting in vascular permeability changes and oedema formation. A variety of methods have been described to evaluate burn-induced plasma extravasation. Most of these methods are based on invasive techniques utilising colloid dyes and radioactive tracers [20±22], tissue biopsies [23] or wet/dry weight ratio [24]. All of these methods require that the experimental tissue is removed, consequently not allowing repetitive measurements in the same animal during the course of time. A non-invasive standardised method may therefore prove valuable for the study of the course of burn-induced tissue oedema and the evaluation of burn treatments aiming at reducing ¯uid losses. 0010-4825/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 1 0 - 4 8 2 5 ( 9 7 ) 0 0 0 3 8 - 3
154
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
Computerised image analysis is currently being used in several ®elds of medical research and examinations. The use of photographic analysis for non-invasive diagnosis of burn depth is described as early as 1977 [25]. A technique of using image analysis for measurements of erythema and blood ¯ow has recently been described by Mattsson et al. [26]. Computerised image analysis has also been employed in clinical follow-up of burn patients [27] and for ¯uorescence digital microscopic evaluation of macromolecular diusion in burn injury [28, 29]. The aims of the present study were to elaborate and evaluate a non-invasive method that allowed continuous or near continuous, objective measurements of extravasated plasma albumin marked with Evans blue in a tissue exposed to a standardised burn injury, and to establish whether the obtained values from image feature analysis correlate with invasive measurements from spectrophotometric analysis of extravasated Evans blue albumin.
2. Materials and methods 2.1. Animals and materials Experiments were performed on 38 male Sprague-Dawley rats weighing 330±340 g. The protocols were approved by the regional Animal Care Committee. Animals were housed for at least 7 days prior to the experiments in a ventilated and temperature-controlled room and had water available ad libitum. The day±night cycle was constant at 12 h light and 12 h dark. Anaesthesia was induced with pentobarbital (50 mg/kg) intraperitoneally and maintained by a continuous intravenous infusion of chloralose (1.5 mg kgÿ1 minÿ1). A tracheostomy was performed and a tracheal cannula was inserted to secure free airway. Blood pressure was monitored using a pressure transducer (Statham P23 AC) connected to a cannula in a femoral artery. Heart rate was measured using a heart rate meter. Body temperature was kept at 388C by placing the animal on a thermoregulated heating pad. 2.2. Thermal trauma model Full-thickness, second degree burn injury was induced in the closely shaved abdominal skin by a technique previously described by Cassuto et al. [22] using an electrically heated aluminium rod with a bottom surface of 1 1 cm connected to an adjustable transformer. A thermosensor electrode from the aluminium rod was connected to a chartwriter (Metrawatt SE 120, ABB, Switzerland) for temperature registration. In order to calibrate the system, room temperature was set on the chartwriter against a thermometer. Room temperature was in the range of 21±238C and was used as the ®rst ®x point on the calibration curve. The bottom surface of the thermoprobe was subsequently sunk into water (5 ml) and heated by turning on the current until the water boiled. At this point the calibration curve levelled o and the level for 1008C was set on the chartwriter, serving as the second ®x point on the calibration curve. Current was cut o and the hot plate was removed from the water, dried, and allowed to cool in the air. When the temperature reached 558C the probe was put in contact with the abdominal skin until the temperature reached 458C, at which time the probe was removed. This procedure allowed a constant amount of heat to be administered to each animal
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
155
independent of possible variations in skin temperature. Probe pressure was standardised to 1.2 N/m2 by pushing down a cylindrical handle compressing in turn a spring on the probe. Evans blue dye (20 mg/kg dissolved in 1 ml of isotonic saline) was administered intravenously to 30 animals 60 min after the burn. At the end of the experiments (180 min) the animals were killed by an intravenous injection of 1 ml saturated KCl. The thermally injured skin area (1 1 cm full thickness burn injury) of Evans blue-treated rats was dissected fullskin, dried on ®lter paper to remove excess ¯uid, and immediately weighed. Each sample was placed in 4 ml formamide (HCONH2) and incubated for 24 h in a water bath at 508C as described by Gamse et al. [30]. Colorimetric measurements were performed on the ¯uid in a Stasar1 (Gilford, U.S.A.) spectrophotometer at the peak absorption of 612 nm. Three measurements were made on each sample and the mean value was used for calculations based on external standards in formamide. 2.3. Fluorescence microscopy preparation The experimental area of four burned animals and the corresponding skin area of four nonburned control animals were prepared for ¯uorescence microscopy as described previously [31]. Evans blue dye was injected intravenously 60 min after the thermal trauma. Two hours later the experimental area was dissected fullskin and the specimens were ®xed in 4% buered formalin for 24 h. Following this procedure the tissue specimens were frozen in liquid nitrogen, sectioned into 10 mm slices in a cryostat (Kryostat 1720, Leitz), mounted on glass slides and dried by heating to 308C for 1 min. The sections were examined in a ¯uorescence microscope (Microphot-FX, Nikon, lens Fluor 10 , ®lter ND 4) using an epi-illumination technique (Ploek Pam System) consisting of a UV-lamp, an excitation ®lter (450±490 nm), a beamsplitting mirror (510 nm) and a suppression ®lter (515 nm). Photographs were taken using ASA 160 ®lm (Kodak Ektachrome 135-36). All specimen analysis was performed by an assistant blinded to the experimental protocol. 2.4. Photographic technique and equipment The technical equipment used for the photographic documentation consisted of a 35 mm single lens re¯ex camera (Contax 167 MT) with state-of-the-art electronic apparatus, except for the automatic focusing device. The camera was provided with a data back for the printing of the exact time of photography on each colour slide. Using a 100 mm macro lens (Carl Zeiss Makro-Planar T, f 2.8) and a telephoto converter (Carl Zeiss Mutar T1 2) a 200 mm optical system with comfortable working distance was achieved. A double electronic ¯ash unit (Combi¯ash, guide number 50 at 100 ISO) was used. Through-The-Lens (TTL) electronic system of the camera limited the exposure deviations. An area of the skin adjacent to and at least 20 mm from the burn area was included in all photographs. Two photographs were taken at each interval with 1 min between in order to evaluate possible exposure deviations. The technical devices and procedures for standardised clinical macrophotography have been described in detail by Eliasson and Heyden [32]. Photographs of
156
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
the shaved abdominal skin were taken immediately before inducing the burn injury (referred as 0 min) and then at 5, 60, 65, 90, 120, 150 and 180 min after the burn trauma. A 36 exposure slide ®lm (Agfachrome 200 RS Professional) was used for the photographs. The ®lms were developed and mounted in plastic frames at a standard ®lm laboratory (Agfachrome Service, Herrljunga, Sweden). Prior to inducing the burn injury, the area where the thermal probe was to be put in contact with the skin was identi®ed and marked with ink. This was done in order to facilitate the identi®cation of the area where image features were to be extracted in the digitised images.
2.5. Image digitisation The colour slides were digitised using an image scanner (Dixel 2000, Hasselblad Electronic Imaging AB, GoÈteborg, Sweden). The digitised images had a resolution of 512 368 pixels with 24 bits per pixel and 8 bits per colour separation. Each pixel had Red (R), Green (G) and Blue (B) values on a scale from 0 to 255. The digital information was then transmitted to an IBM compatible PC (Victor 486 DX) with an extended graphics card (AT Vista Truevision Inc., Indianapolis, U.S.A.) for image feature extraction and analysis. The software designed for this purpose was developed in collaboration with Hasselblad Electronic Imaging AB (GoÈteborg, Sweden).
2.6. Image feature extraction The border of the burn area was identi®ed and outlined and an area of approximately 10 000 pixels was created (Fig. 1). The location and co-ordinates of each created object in each slide was stored on the hard disc of the computer. The same object was then used and positioned in the corresponding burn area on subsequent slides taken.
Fig. 1. Clinical appearance of rat skin prior to thermal injury (left) and 180 min postburn (right), i.e. 120 min after IV administration of Evans blue dye. The areas where image features were extracted in the digitised images are framed.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
157
2.7. Colour systems Two dierent colour systems were used in the image feature analysis. The purpose was to evaluate whether any of the systems presented advantages for the biological interpretation of the observed colour dierences, but also to establish whether correlation existed between two dierent colour features and data obtained by spectrophotometric analysis of extravasated Evans blue albumin. The colour systems used were:
2.7.1. Normalised red (n-r), green (n-g) and blue (n-b) values (rgb) The colour system describes the relative amount of Red (R), Green (G), and Blue (B) respectively for each pixel. R 0Rn ÿ rR255 n ÿ r 255 RGB
Fig. 2. Appearance of rat skin prior to thermal injury (upper diagrams) and at 120 min after administration of Evans blue dye (lower diagrams). The distribution of mean values for normalised-red, -green, -blue and Intensity, Hue and Saturation values from left to right in the marked areas are demonstrated. Normalised rgb (left): Note the increase in normalised-blue value and the decrease in normalised-red value in the burned area with extravasated Evans blue. Intensity-Hue-Saturation (right): The blue discoloration in the thermally injured area is demonstrated by an increase of Saturation value and a decrease of Hue and Intensity values when compared to values prior to thermal injury.
158
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
n ÿ g 255
G RGB
n ÿ b 255
B RGB
0Rn ÿ gR255 0Rn ÿ bR255
For example, an area with a high normalised blue (n-b) value and low normalised red (n-r) and green (n-g) values is visually perceived as a blue colour (Fig. 2, lower left diagram).
2.7.2. Hue(H)-Saturation(S)-Intensity(I) values (HSI) The colour system is built on perceptive parameters. The distribution of colours is shown in Fig. 3. The hue parameter describes the colour, for example, red or blue. The colours are arranged in a radial pattern and each hue can consequently have a value ranging from 0 to 360 degrees. Intensity is a colour-neutral parameter that describes the relative lightness or darkness of the hue. Saturation shows how much the hue is mixed with white. A highly saturated colour contains very little white and the saturation consequently increases towards the periphery of the circle. The values for Intensity-Hue-Saturation were calculated as follows (modi®ed from Ledley [33]). Hue p GÿB 180 0RH<360 H Arc tan 3 2R ÿ G ÿ B Saturation S 1 ÿ 3 minr,g,b 255 0RSR255 [r,g,b] means the value for one of n-r, n-g or n-b.
Fig. 3. Distribution of Hue values in the HSI-colour system. Note the location of the red and blue colours. The Saturation increases towards the periphery of the circle.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
159
Intensity I
RGB 0RIR255 3
Mean values and standard deviations for the colour features described above were measured within each created object and stored in a separate ®le for subsequent statistical analysis. Tissue colour changes were expressed as the dierence in mean values for the colour parameters from one interval to the next. The changes in HSI values due to colour changes are shown in Fig. 2 (right diagrams). 2.8. Comparison of the two colour systems A comparative analysis of the two colour systems was performed in order to establish whether linear correlation existed between the changes observed for the image parameters in order to interpret the clinical impression of colour changes. 2.9. Statistical analysis Paired two-tailed Student's t-test was used in the statistical analysis and a p-value less than 0.05 was regarded as statistically signi®cant. The comparison between image features and spectrophotometric values was performed by linear correlation analysis.
3. Results 3.1. General One animal died during the ®rst hour of the experiments. Two tissue specimens subject to spectrophotometric analysis were lost due to identi®cation problems, leaving 27 samples for the correlation analysis. No cardiovascular eects of the burn trauma or the administration of Evans blue were observed during the experiments. 3.2. Fluorescence histology Skin specimens from four burned animals subject to ¯uorescence histology demonstrated rich Evans blue ¯uorescence in all layers of the skin, whereas in four non-burned control animals the ¯uorescence was mainly localised in the lumen and walls of blood vessels. 3.3. Spectrophotometry Administration of Evans blue 60 min after the thermal trauma resulted in a signi®cant blue coloration of the skin at 180 min postburn. The amount of Evans blue in the skin was 59.129.3 ng/mg tissue (mean2SEM, n = 27).
160
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
3.4. Normalised rgb values The colour changes during the course of the experiment are shown in Fig. 4. During the interval 0±5 min only minor and statistically not signi®cant changes were recorded for the 29 animals. From 5 to 60 min, small but statistically signi®cant changes were observed. Changes comprised of a small decrease in n-r value and a minor increase in n-b value. At 65 min postburn, i.e. immediately after intravenous administration of Evans blue the colour pattern showed fast increases in n-g and n-b values and a corresponding decrease in n-r value. These changes were statistically signi®cant ( p < 0.001). The major colour dierences were, however, observed in the intervals between 65±90 min and 90±120 min. During these intervals there was a continuous increase in n-b value and decrease in n-r value ( p < 0.001). Between 120 and 150 min the visible changes were smaller but statistically signi®cant for n-r and n-b values ( p < 0.05). In the last interval from 150 to 180 min, the observed changes were not signi®cant. For the group as a whole, the n-g value remained relatively constant during the entire period of observation. 3.5. Hue-Saturation-Intensity As shown in Fig. 5, only minor changes in HSI values were found in the interval from 0 to 5 min. During 5±60 min minor but statistically signi®cant ( p < 0.001) decreases in Hue and Saturation values were detected. From 60 to 65 min, a further decrease in Hue value was observed together with a minor drop in Saturation ( p < 0.001). As mentioned above, the most prominent colour changes in the burn area were observed in the intervals from 65 to 90 min and 90 to 120 min ( p < 0.001). During these periods a continuous decrease in Intensity and Hue values were detected, paralleled by an increase in Saturation values. The changes were of
Fig. 4. The distribution of mean (2SEM) pixel values for normalised-red, -green and -blue (n-r, n-g, n-b) in thermally injured rat skin. Note the changes for n-b values and for n-r values after administration of Evans blue dye at 60 min post burn.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
161
Fig. 5. Changes in the Intensity-Hue-Saturation colour system (mean 2 SEM) after thermal injury to rat skin. Note the changes in all three parameters occurring after administration of Evans blue dye at 60 min post burn.
comparable magnitude in the two intervals. After 120 min only minor and statistically not signi®cant changes could be detected. 3.6. Comparison of the two colour systems Correlation analysis showed that there was a high linear correlation between the changes in normalised rgb values respectively and dierences in Intensity-Hue-Saturation values from 0 to 180 min (Table 1). Decreases in Intensity and Hue values correlated signi®cantly with decreases in n-r and n-g values and an increase in n-b value. An increase in Saturation was correlated to decreased n-r and n-g values and increased n-b value. 3.7. Comparison between spectrophotometry and digital image analysis Correlation analysis of Evans blue spectrophotometry values and of changes in n-b values 60±180 min postburn, showed a high linear correlation coecient of 0.89 ( p < 0.001) (Table 2). 3.8. Methodological errors Image feature values from two consecutive photographs of a corresponding area were compared. The analysis revealed that the dierences between exposures were not signi®cant and had no in¯uence on the interpretation of the results (Table 3). Table 1 Correlation analysis between two colour systems. Results from linear correlation analysis between measured dierences in image feature values from 0 to 180 min for normalised-red, -green and -blue values (n-r, n-g, n-b) and Hue-Saturation-Intensity (HSI) values
Hue Saturation Intensity
n-r
n-g
n-b
0.83 *** ÿ0.77 *** 0.55**
0.37 * ÿ0.71 *** 0.83 ***
ÿ0.80 *** 0.87 *** ÿ0.73 ***
* = p < 0.05, ** = p < 0.01, *** = p < 0.001, n = 27.
162
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
Table 2 Correlation analysis between spectrophotometry and the DIA technique. Correlation coecients and probability values after linear correlation analysis between spectrophotometry values and normalized-red, -green and -blue values (n-r, n-g, n-b) and Intensity-Hue-Saturation values, respectively
Spectrophotometry Spectrophotometry
n-r
n-g
n-b
ÿ0.82 *** Hue ÿ0.59 **
ÿ0.64 *** Saturation 0.85 ***
0.89 *** Intensity ÿ0.69 ***
** = p < 0.01, *** = p < 0.001, n = 27. Table 3 Methodological error values of the DIA technique. Mean dierences in normalised-red, -green and -blue values (n-r, n-g, n-b) and Hue-Saturation-Intensity (HSI) values measured in corresponding areas in two consecutive photographs at the same time interval (n = 29). Mean dierences were not signi®cant Normalised values
n-r
Mean dierence SD SEM
0.20 1.24 0.21
HSI-values Mean dierence SD SEM
Hue 0.12 0.77 0.13
n-g 0.04 0.39 0.07 Saturation 0.71 3.64 0.63
n-b 0.24 1.21 0.21 Intensity 0.30 4.78 0.83
4. Discussion Skin burn injury involves a wide range of vascular changes often leading to increased permeability and subsequent oedema formation [4]. Traditionally, experimental animal models investigating burn-induced oedema have used invasive methods such as tissue wet/dry weight [24, 34], and analysis of Evans blue albumin extravasation by spectrophotometry [22, 35]. A few non-invasive methods, such as dichromatic absorptiometry [23], estimation of lymph ¯ow from burned areas [11, 36, 37] and measurement of rat limb volume [38] have also been described. Invasive methods are generally accepted to be more exact but require that tissue specimens are taken from the animals thereby not allowing continuous evaluation of progressive changes in the burn area of the same animal. Moreover, invasive methods cannot be employed in human subjects, necessitating the development of sensitive non-invasive methods for the study of burn pathophysiology and evaluation of burn treatments. In the present study a non-invasive technique was developed allowing repetitive evaluation of colour changes in an area of thermal injury. Our objective was to decide whether such colour changes were representative of the pathophysiology of experimental burn trauma and if quanti®cation and statistical analysis of physiological and pathophysiological processes could be accurately performed. Results showed that intravenous administration of Evans blue dye resulted in a pronounced blue discoloration of the experimental area (Fig. 1) suggesting a pronounced accumulation of
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
163
extravasated Evans blue albumin. The distribution of Evans blue albumin in the burned area was analyzed by ¯uorescence microscopy and revealed that large amounts of Evans blue had accumulated in all layers of the skin supporting the fact that this is a full-thickness burn. In non-burned control animals, however, Evans blue was mainly located inside the blood vessels, suggesting that the bluish discoloration of the surrounding non-burned skin was representative of intravasal non-extravasated Evans blue albumin. Normal, shaved rat skin contains almost equal amounts of red, green and blue colour, with a slight domination of red (Fig. 2, upper left panel). Repeated photographs of the burned area followed by normalised rgb analysis allowed a near continuous monitoring of EB-albumin extravasation in the burned tissue. Results showed a continuous increase in n-b values and a corresponding decrease in n-r, while n-g remained virtually unchanged with time (Fig. 4). Since normalised rgb values have been used, an isolated increase in the blue colour must result in an equivalent decrease of the sum of the other two colours. These results were in line with colour changes observed by the naked eye showing continuously increased blue coloration of the burned skin. Correlation analysis between values obtained by spectrophotometric quanti®cation of Evans blue in the burned skin and normalised rgb values obtained by the DIA technique showed signi®cant correlation for all tested variables (Table 2). A negative correlation was seen for n-r and n-g due to the decreasing values for these variables as opposed to increasing spectrophotometric values due to increased EB-leakage. Despite small changes in the normalised green values, a signi®cant negative correlation was obtained due to the high sensitivity of the DIA technique. Since both n-b and spectrophotometric values increased towards the end of the experiment, a positive correlation was found (Table 2). The dierence in n-b values between the time of administration of Evans blue (60 min postburn) and the end of the experiment (180 min postburn) can thus be used as a quantitative expression of oedema formation in the same way as spectrophotometry analysis of accumulated extravasated plasma albumin in the experimental skin area. However, since spectrophotometric analysis in this study is performed on the full-skin preparation, data are representative of the total content of Evans blue albumin in the burned skin tissue. The question is whether colour changes observed by the DIA technique are representative of changes in all skin layers or only in the skin surface. Rat skin is rather translucent, since subcutaneously located blood vessels can be observed by the naked eye, suggesting that the colour changes monitored by the DIA technique are probably representative of colour changes in the full thickness of the skin. The same colour changes could also be expressed using the HSI system (Fig. 5). Evans blue accumulation in the burned tissue resulted in decreased Intensity values in the burned area corresponding with an observed darkening of the experimental area (Fig. 5). In normal rat skin, the Hue value lies near 1808, i.e. close to the red colour (Fig. 3). In connection with increased blue coloration of the burned area the Hue value decreased towards 608 and, as expected, ending in the blue sector. Saturation is inversely related to the amount of white colour. In normal rat skin the Saturation values are low since the skin appears whitish. This relationship leads to increased Saturation values due to decreased amount of white following accumulation of blue colour (Fig. 5). The blue discoloration could be expressed as a continuous decrease in Intensity and Hue values concomitant with an increase in Saturation values. All of these changes signi®cantly correlated with spectrophotometric values (Table 2).
164
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
These data suggest that Saturation values and the inversed values of Intensity or Hue can independently be used as quantitative variables describing the continuous accumulation of Evans blue albumin in the burned skin. The methodological errors associated with the DIA technique were limited and did not have any signi®cant in¯uence on the results when expressed as the standard deviation (SD) between the values obtained in two consecutive exposures (Table 3). The camera equipment used for photographic registration combined with an image scanner which converted the slides into digital images demonstrated a reproducibility which was adequate and sucient for the experimental purposes of the present study, as suggested by low mean dierences (less than 5%; Table 3). Since the DIA technique is based upon analysis of visible colour in the skin, it is essential to shave as close to the skin as possible in order to reduce the disturbing eects of shading. It must be stressed that during preparation of the animals care must be taken not to mechanically traumatise the skin since it would jeopardise the possibility of performing adequate measurements with the digital technique. In conclusion, both normalised rgb values and HSI values showed signi®cant correlation with spectrophotometric values (Table 2). These results suggest that the non-invasive DIA technique can be used as an alternative to invasive spectrophotometric analysis in quantifying Evans blue albumin accumulation in burned tissue. The DIA technique is, however, superior to invasive methods by allowing repeated measurements in the same area without interfering with the pathophysiological processes. In addition, correlation analysis between the two colour systems showed a signi®cant correlation between normalised rgb values and HSI values, suggesting that both systems can be used independently for repetitive monitoring of tissue colour changes.
5. Summary Profuse oedema formation is one of the main characteristics of skin burn. Most methods used to quantify experimental burn oedema are based on invasive techniques which do not allow repeated measurements in the same tissue. The present study evaluated a non-invasive method based on digital image analysis that allows near-continuous quanti®cation of extravasated Evans blue albumin in a standardised burn model. A standardised 1 cm2 full-thickness 2nd degree burn injury was induced in the abdominal skin of 30 anaesthetised rats. One hour post-burn the animals received a single injection of Evans blue (EB) dye. Two hours after the EB-injection, the burn area was dissected full skin and its contents of Evans blue were quanti®ed using spectrophotometry. Colour slides of the burn area were digitised and two dierent colour-systems (normalised-rgb values and the HueSaturation-Intensity system) were used to quantify skin contents of Evans blue and correlated to spectrophotometric data. Correlation coecients for spectrophotometry versus normalised blue were 0.89 ( p < 0.001), normalised red ÿ0.82 ( p < 0.001) and normalised green were ÿ0.64 ( p < 0.001), respectively. Correlation coecients for Hue-Saturation-Intensity system vs spectrophotometry were: Hue ÿ0.59 ( p < 0.01), Saturation 0.85 ( p < 0.001), Intensity ÿ0.69 ( p < 0.001). Methodological errors were negligible.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
165
We have developed a non-invasive method for quantifying experimental burn oedema. The present non-invasive technique for quantifying experimental burn oedema demonstrated a good correlation when compared to invasive spectrophotometry, but is superior by allowing near continuous measurement in the same subject and may be of value for evaluation of eective burn oedema reducing treatment.
Acknowledgements This work was supported by grants from the Bohus County Council and the Medical Society of GoÈteborg.
References [1] J.V. Boykin Jr.., E. Eriksson, M.M. Sholley, R.N. Pittman, Histamine-mediated delayed permeability response after scald burn inhibited by cimetidine or cold-water treatment, Science 209 (1980) 815. [2] J.V. Boykin Jr.., S.L. Crute, Inhibition of increased serum histamine and lactate after severe scald injury and cold-water treatment, Curr. Surg. 38 (1981) 393. [3] J.V. Boykin Jr.., E. Eriksson, M.M. Sholley, R.N. Pittman, Cold-water treatment of scald injury and inhibition of histamine-mediated burn edema, J. Surg. Res. 31 (1981) 111. [4] W.G. Cio Jr.., J.E. DeMeules, R.L. Gamelli, Vascular reactivity following thermal injury, Circ. Shock 25 (1988) 309. [5] M.S. Starr, G.B. West, Bradykinin and oedema formation in heated paws of rats, Br. J. Pharmacol. 31 (1967) 178. [6] G. Arturson, M. Hamberg, C.-E. Jonsson, Prostaglandins in human burn blister ¯uid, Acta Physiol. Scand. 87 (1973) 270. [7] D.N. Herndon, S. Abston, M.D. Stein, Increased thromboxane B2 levels in the plasma of burned and septic burned patients, Surg. Gynecol. Obstet. 159 (1984) 210. [8] R.H. Demling, C. LaLonde, Topical ibuprofen decreases early postburn edema, Surgery 102 (1987) 857. [9] T.J. Williams, Prostaglandin E2, prostaglandin I2 and the vascular changes of in¯ammation, Br. J. Pharmacol. 65 (1979) 517. [10] J.P. Heggers, F. Ko, M.C. Robson, R. Heggers, K.E. Craft, Evaluation of burn blister ¯uid, Plast. Reconstr. Surg. 65 (1980) 798. [11] C.-E. Jonsson, Y. Shimizu, B.B. Fredholm, E. GranstroÈm, E. Oliw, Eux of cyclic AMP, prostaglandin E2 and F2a and thromboxane B2 in leg lymph of rabbits after scalding injury, Acta Physiol. Scand. 107 (1979) 377. [12] J.P. Heggers, G.L. Loy, M.C. Robson, E.J. Del Beccaro, Histological demonstrations of prostaglandins and thromboxanes in burned tissues, J. Surg. Res. 28 (1980) 110. [13] C. Denzlinger, S. Rapp, W. Hagmann, D. Keppler, Leukotrienes as mediators in tissue trauma, Science 230 (1985) 330. [14] F. Alexander, M. Mathieson, K.H.T. Teoh et al, Arachidonic acid metabolites mediate early burn edema, J. Trauma 24 (1984) 709. [15] W.J. Schirmer, J.M. Schirmer, G.B. Na, D.E. Fry, Complement-mediated hemodynamic depression in the early postburn period, J. Trauma 29 (1989) 932. [16] K.T. Oldham, K.S. Guice, G.O. Till, P.A. Ward, Activation of complement by hydroxyl radical in thermal injury, Surgery 104 (1988) 272. [17] K.T. Oldham, K.S. Guice, G.O. Till, P.A. Ward, Evidence of local complement activation in cutaneous thermal injury in rats, Prog. Clin. Biol. Res. 264 (1988) 421.
166
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
[18] P.A. Ward, G.O. Till, Pathophysiologic events related to thermal injury of skin, J. Trauma 30 (Suppl.) (1990) S75. [19] E.T. Wei, J.G. Kiang, Peptides of the corticoliberin superfamily attenuate thermal and neurogenic in¯ammation in rat pawskin, Eur. J. Pharmacol. 168 (1989) 81. [20] W.W.L. Glenn, J. Muus, C.K. Drinker, Observations on the physiology and biochemistry of quantitative burns, J. Clin. Invest. 22 (1943) 451. [21] O. Cope, F.D. Moore, A study of capillary permeability in experimental burns and burn shock using radioactive dyes in blood and lymph, J. Clin. Invest. 23 (1944) 241. [22] J. Cassuto, P. NellgaÊrd, L. Stage, A. JoÈnsson, Amide local anesthetics reduce albumin extravasation in burn injuries, Anesthesiology 72 (1990) 302. [23] R.H. Demling, R.B. Mazess, R.M. Witt, W.H. Wolberg, The study of burn edema using dichromatic absorptiometry, J. Trauma 18 (1978) 124. [24] H. Onarheim, T. Lund, R. Reed, Thermal skin injury: II. Eects on edema formation and albumin extravasation of ¯uid resuscitation with lactated Ringer's, plasma, and hypertonic saline (2400 mosmol/l) in the rat, Circ. Shock 27 (1989) 25. [25] V.J. Anselmo, B.E. Zawacki, Multispectral photographic analysis. A new quantitative tool to assist in the early diagnosis of thermal burn depth, Ann. Biomed. Eng. 5 (1977) 179. [26] U. Mattsson, A. JoÈnsson, M. Jontell, J. Cassuto, Digital image analysis (DIA) of colour changes in human skin exposed to standardized thermal injury and comparison with laser doppler measurements, Comput. Methods. Programs Biomed. 50 (1996) 31. [27] D. Franceschi, R.L. Gerding, R.B. Fratianne, Microcomputer image processing for burn patients, J. Burn Care Rehabil. 10 (1989) 546. [28] S.J. Aggarwal, S.J. Shah, K.R. Diller, C.R. Baxter, Fluorescence digital microscopy of interstitial macromolecular diusion in burn injury, Comput. Biol. Med. 19 (1989) 245. [29] S.J. Aggarwal, K.R. Diller, H.K. Yeung, C.R. Baxter, Local macromolecular extravasation in thermal burns quanti®ed by ¯uorescent video microscopy and computer vision, J. Burn Care Rehabil. 15 (1994) 104. [30] R. Gamse, P. Holzer, F. Lembeck, Decrease of substance P in primary aerent neurones and impairment of neurogenic plasma extravasation by capsaicin, Br. J. Pharmacol. 68 (1980) 207. [31] A. Saria, J.M. Lundberg, Evans blue ¯uoroscence: quantitative and morphological evaluation of vascular permeablility in animal tissues, J. Neurosci. Methods 8 (1983) 41. [32] L. Eliasson, G. Heyden, Clinical macrophotography for oral health monitoring purposes. Methods and results from a comparative study, Swed. Dent. J. 14 (1990) 1. [33] Ledley, R.S., Buas, M., Golab, T.J., 1990. Fundamentals of true-color image processing. In: IEEE (Ed.), 10th International Conference on Pattern Recognition, Atlantic City, U.S.A., vol. 1, p. 791. [34] I. Blomgren, U. Bagge, Postburn blood ¯ow, edema, and survival of the hairy mouse ear after scald injury at dierent temperatures, Scand. J. Plast. Reconstr. Surg. 18 (1984) 269. [35] A. Saria, Substance P in sensory nerve ®bres contributes to the development of oedema in the rat hind paw after thermal injury, Br. J. Pharmacol. 82 (1984) 217. [36] G. Arturson, C.-E. Jonsson, Eects of indomethacin on the transcapillary leakage of macromolecules and the eux of prostaglandins in the paw lymph following experimental scalding injury, Ups. J. Med. Sci. 78 (1973) 181. [37] J.R. Casley-Smith, J.R. Casley-Smith, The eects of diosmin (a benzo-pyrone) upon some high-protein oedemas: lung contusion, and burn lymfoedema of rat legs, Agents Actions 17 (1985) 14. [38] G. Arturson, O.P. Jakobsson, Oedema measurements in a standard burn model, Burns Incl. Therm. Inj. 12 (1985) 1.
Anders JoÈnsson MD PhD is sta specialist at the Department of Orthopedic Surgery, Sahlgrenska University Hospital, MoÈlndal, Sweden and research fellow at the Department of Physiology, GoÈteborg University, GoÈteborg, Sweden. His research interest is the pathophysiology of trauma response and especially the response to thermal trauma.
A. JoÈnsson et al. / Computers in Biology and Medicine 28 (1998) 153±167
167
Ulf Mattsson DDS PhD works at the Clinic of Oral and Maxillofacial Surgery and Hospital Dental Care, Central Hospital, Karlstad, Sweden. His research has been focused on elaborating non-invasive methods for evaluation of skin and oral mucosal disorders. He is currently involved in research in the ®eld of Oral Medicine with special emphasis on pathogenesis and treatment of oral mucosal lesions. Jean Cassuto MD PhD is Research Director at Sahlgrenska University Hospital, MoÈlndal. He is also leading a research group at the hospital on several research areas including burns, pain, and in¯ammatory mechanisms. In addition to clinical research, the group is conducting experimental research at the Department of Physiology, GoÈteborg University. Dr Cassuto has been the tutor of eleven thesis-projects, eight of which have been presented at GoÈteborg University, Sweden. Guy Heyden OD MSc is Professor in Oral Pathology, UNESCO-Cousteau Ecotechnie Chairholder and Dean, Faculty of Thematic Studies, GoÈteborg University, Sweden. As Supervisor of the interdisciplinary Koster Health Project, Guy Heyden has developed standardized photographic methods for the monitoring of human health changes and neighbouring environmental variationsÐroutines facilitating retrospective studies utilizing, for example, digital image analysis.