Br. J. exp. Path. (1975) 56, 83
THE RESOLUTION OF THERMAL OEDEMA AT VARIOUS TEMPERATURES UNDER COUMARIN TREATMENT N. B. PILLER From the Department of Zoology, University of Adelaide Received for publication 30 September 1974
Summary.-The treatment of thermal oedema with coumarin (a benzopyrone) has been found to be most effective when fast moving air of 200 was blown over the burnt animals. Coumarin has two main effects: one is to cause vascular injury thus allowing extra protein and fluid into the tissues; the other is to stimulate phagocytosis, enzyme production and thus proteolysis and a subsequent removal of protein and fluid from the injured tissues. At lower temperatures the injurious nature of coumarin is prominent. At medium range temperatures the proteolytic actions of coumarin outweigh its injurious nature. Resolution thus proceeds much more rapidly. At high temperatures resolution is slowed. This is a consequence of the antioxidant effect of coumarin on adrenaline and ambient temperature on peripheral dilatation. The results obtained tie in well with those obtained by workers using water of equivalent temperature. A GROUP of drugs called benzopyrones have been shown to reduce high protein oedemas in animals and man (CasleySmith and Piller, 1974a,b; F6ldi-Borcsok, 1972; Foldi-Borcs6k and Foldi, 1973; Piller, 1974b). However, there has been confusion as to the mode of action of this group of drugs, which includes coumarin, the drug dealt with in this paper. It is now becoming apparent that they have two major effects. Firstly, they cause injury to the vascular endothelium (Lecomte, van Cauwenberge and Lallemand, 1971). This has also been confirmed in our laboratory (Casley-Smith and Piller, 1974a,b; Piller, 1974a). Although Lecomte did not distinguish between the effects of the benzopyrones tested on the various vessels, results from our laboratory (Piller, 1974a) show that generally they prevent increases in permeability which in their absence would have occurred, while they cause additional injury to vessels which are normally unaffected. Additional evidence for this injurious nature of the drugs comes from an electron microscopical study by Casley-Smith, Foldi-Borcsok
and Foldi (1974) which showed that troxerutin and Venalot (which contains coumarin and sodium rutin sulphate) cause additional numbers of open junctions. Such blood vascular injury, although releasing extra protein and fluid into the tissues (Piller, 1 974a), is more than compensated for by the proteolytic actions of the benzopyrones. They cause proteolysis of the abnormal accumulated protein in the extracellular extrafibrilar compartment (Casley-Smith and Piller, 1974a,b; Piller and Casley-Smith, 1974). This allows a net removal of protein (Piller, 1974d). There has also been resurging interest in the use of cold water treatment of the high protein oedema of burn injury (King and Zimmerman, 1965; Ofeigsson, 1965; Blumenfield, 1974). However, there are great differences in the temperatures suggested to promote the maximal resolution and minimal discomfort in thermal injury. King and Zimmerman (1965) suggested that surface cooling at 10-140 should be applied as soon as possible after burn injury and continued for 30 min. However, Ofeigsson (1965) showed that
84
N. B. PILLER
very cold water (-. 00) greatly raised the mortality of burnt animals, indeed even water at 15 or 180 still resulted in an increased mortality. Ofeigsson suggested that the best way of treating burns was by an initial immersion at 22-25° for 2-5 min followed by longer immersion in 30° water. Shulman (1960) suggested ice water treatment at 5-13° from 30 min to 5 h, depending on the severity of the injury. Wilson et al. (1963) found water at 16° for 15 min to 1 h to be most useful in the treatment of hot (63°) water burns. Despite these variations, it cannot be denied that cold water treatment of all types of thermal injury provides a quick and readily available therapy. However, what if the cold water cannot be applied immediately? King and Zimmerman (1965) have shown that 80-85% of the oedema which follows burn injury developed in the first 30 min although small amounts continued to form until the end of the third hour, but Harkins (1942, 1944) claims " it only completely subsides after 48 hours ". Thus, if cold is not applied immediately some oedema will still result. This is where the administration of the benzopyrones can be very important. The protein which leaks through the injured endothelium and enters the tissues (where it attracts oedema fluid) can be removed by proteolysis. This allows the small fragments to leave the tissues via the closed junctions of the blood vessels (Casley-Smith and Piller, 1974a,b; Piller, 1974a). The removal of protein frees the oedema fluid, with a resultant rapid resolution of the oedema. Since both cold and benzopyrone therapy are very effective in reducing burn oedema when given alone, the question was raised would they be better if given in combination? To date, there is no literature on the effectiveness of coumarin in the resolution of thermally induced oedema at temperatures other than those which must be assumed to be room temperature (F6ldiBorcsok et al., 1972; Casley-Smith et al., 1974; Casley-Smith and Piller, 1974a,b).
The amount of fluid loss into thermally injured regions, and thus the amount of oedema, is strongly dependent on blood flow and this on temperature (Courtice, 1946; Abramson, 1967). Thus, by keeping the animal cool, the outpouring of plasma into the injured regions can be reduced (Harkins, 1942, 1944; King and Zimmerman, 1965). Courtice (1946) reported a reduction in lymph flow by as much as 500% under cold treatment. The opposite has been observed as a consequence of the application of heat (Blalock and Baltimore, 1942). Thus, for the present experiments temperatures ranging from 10 to 350 with and without coumarin were used. Although it is well known that the lower range of temperatures used the lower tissue metabolic rate (Stone and Martin, 1958), it has no effect on the healing process of thermally injured limbs (Courtice, 1946). High temperatures were also tested although these were expected to be deleterious in aiding the resolution of thermal oedema (Blalock and Baltimore, 1942). The highest temperature of 350 was used in the light of a paper by Grayson (1949), in which it was shown temperatures of 36-40' caused the vasoconstriction of cutaneous vessels after their initial dilatation. It was thought that the vasodilatatory phase when combined with coumarin therapy might initially allow more rapid entry of the drug to the thermally injured regions, while the phase associated with vasoconstriction could possibly reduce the outpouring of protein and fluid from these vessels into the tissues. The net result might be a much more rapid rate of resolution. MATERIALS AND METHODS FoI the experiment 80 female albino rats, weight 200 ± 10 g, were used. Trwo days before the experiment both rear hind legs of all rats were closely shaven with an electric razor. They were then divided into 2 groups, each of which was then divided into 4 subgroups, 2 of which received intraperitoneal coumarin (25 mg/kg body weight in a 2% solution of A. R. ethyl alcohol in distilled water) immediately before the administration of bturn injtury. The other 2
85
TREATMENT OF THERMAL OEDEMA WITH COUMARIN
thermally induced oedema, they were of no use when this type of oedema was treated with coumarin at various ambient temperatures. Thus, a much simpler model was used. This consisted of 2 regression lines: one extending from zero time to the maximal recorded volumes and the other from this volume to the end of the observations. In this calculation, the values for each animal of each group were considered.
subgroups were given an equivalent volume of 2% A. R. ethyl alcohol in distilled water. All members of all subgroups were then given ether anaesthesia. Following this, the right hind limbs of all members of one group were burnt. For this the limbs were immersed in hot flowing water of 540 for 60 s. Temperature was maintained to within ± 0 5°. The injured limbs were then gently blotted dry with soft towelling. Following recovery from anaesthesia (5 min, at which stage there was no observable swelling on the injured limbs) the burnt rats with or without the coumarin therapy, were placed in wire cages in forced draught ventilated temperature cabinets, either at 10, 20, 30 or 35°. Adequate food and water were supplied. Simultaneously, all members of the other group were distributed to the various temperature cabinets. No members of this group were burnt. Two of the subgroups received coumarin while the others acted as a control, as before. Thus, at each temperature level there were 4 subgroups, viz., burn, burn with coumarin, normal (unburnt-untreated) and normal with coumarin. Measurements of both hind paws to the ankle joints were then performed by plethysmographic methods as described previously (Piller, 1974b). The times of measurement were immediately preceding thermal injury or entry into the temperature cabinets and at 3, 6, 7, 9, 10, 12, 16, 24, 48, 72, 130 and 200 h following injury or at the equivalent times for those groups without thermal injury. For each animal, corrections were made to the leg volumes to allow for the effect of temperature alone, both on the injured legs and on the normal ones. Oh-ishi and Sakuma (1970a, b) have suggested a number of simple models for rat paw oedema. Although these were claimed to fit
RESULTS
To ascertain the effectiveness of temperature with and without coumarin on thermally induced oedema, 5 parameters were examined. These were (1) the rate of swelling of the oedematous leg, (2) the maximal volume attained by the leg during the period of observation, (3) the time at which this maximal swelling was reached, (4) the rate of resolution of the oedema from the time of maximal swelling and (5) the time taken for the injured leg to return to normal.
1. Rate of swelling (Table I) In all cases from 10-35° the treatment of the oedema with coumarin increased the rate of swelling of the injured limb. However, the increase was only significant at 200 (0 001 < P < 0 01) and 350 (0 01< P < 0.05).
TABLE I.-Effect of Varying Temperatures ± Coumarin on the Rate of Swelling of Thermally Injured Limbs. Temp. 100 100 200 200
Treatment
Burn' Burn + coumarin2 Burn Burn + coumarin Burn Burn + coumarin
No. 5 5 5 5 5 5 5 5
Rate of swelling (ml/h) (X 10-2) 2-8
4.9
S.E. (X 10-3)
p3
8.3 10-2 8-6 12-0 5-3 7 9 10-5 10-0
4-3 ft 7-3 300 1-8 300 2-5 350 Burn 1-8 t 350 Burn + coumarin 5-8 1 Burn given by immersion of leg in hot (540), moving water for 1 min. 2 Coumarin, freshly prepared was administered i.p. at 25 mg/kg body weight in a 2 % solution of A.R. ethyl alcohol in physiological saline. Control groups received same volume of a 2 % solution of A.R. ethyl alcohol in saline. 3 Arrows
indicate direction and significance of change in oedema swelling -P>0 05; t 0 01
THE RESOLUTION OF THERMAL OEDEMA AT VARIOUS TEMPERATURES UNDER COUMARIN TREATMENT N. B. PILLER From the Department of Zoology, University of Adelaide Received for publication 30 September 1974
Summary.-The treatment of thermal oedema with coumarin (a benzopyrone) has been found to be most effective when fast moving air of 200 was blown over the burnt animals. Coumarin has two main effects: one is to cause vascular injury thus allowing extra protein and fluid into the tissues; the other is to stimulate phagocytosis, enzyme production and thus proteolysis and a subsequent removal of protein and fluid from the injured tissues. At lower temperatures the injurious nature of coumarin is prominent. At medium range temperatures the proteolytic actions of coumarin outweigh its injurious nature. Resolution thus proceeds much more rapidly. At high temperatures resolution is slowed. This is a consequence of the antioxidant effect of coumarin on adrenaline and ambient temperature on peripheral dilatation. The results obtained tie in well with those obtained by workers using water of equivalent temperature. A GROUP of drugs called benzopyrones have been shown to reduce high protein oedemas in animals and man (CasleySmith and Piller, 1974a,b; F6ldi-Borcsok, 1972; Foldi-Borcs6k and Foldi, 1973; Piller, 1974b). However, there has been confusion as to the mode of action of this group of drugs, which includes coumarin, the drug dealt with in this paper. It is now becoming apparent that they have two major effects. Firstly, they cause injury to the vascular endothelium (Lecomte, van Cauwenberge and Lallemand, 1971). This has also been confirmed in our laboratory (Casley-Smith and Piller, 1974a,b; Piller, 1974a). Although Lecomte did not distinguish between the effects of the benzopyrones tested on the various vessels, results from our laboratory (Piller, 1974a) show that generally they prevent increases in permeability which in their absence would have occurred, while they cause additional injury to vessels which are normally unaffected. Additional evidence for this injurious nature of the drugs comes from an electron microscopical study by Casley-Smith, Foldi-Borcsok
and Foldi (1974) which showed that troxerutin and Venalot (which contains coumarin and sodium rutin sulphate) cause additional numbers of open junctions. Such blood vascular injury, although releasing extra protein and fluid into the tissues (Piller, 1 974a), is more than compensated for by the proteolytic actions of the benzopyrones. They cause proteolysis of the abnormal accumulated protein in the extracellular extrafibrilar compartment (Casley-Smith and Piller, 1974a,b; Piller and Casley-Smith, 1974). This allows a net removal of protein (Piller, 1974d). There has also been resurging interest in the use of cold water treatment of the high protein oedema of burn injury (King and Zimmerman, 1965; Ofeigsson, 1965; Blumenfield, 1974). However, there are great differences in the temperatures suggested to promote the maximal resolution and minimal discomfort in thermal injury. King and Zimmerman (1965) suggested that surface cooling at 10-140 should be applied as soon as possible after burn injury and continued for 30 min. However, Ofeigsson (1965) showed that
84
N. B. PILLER
very cold water (-. 00) greatly raised the mortality of burnt animals, indeed even water at 15 or 180 still resulted in an increased mortality. Ofeigsson suggested that the best way of treating burns was by an initial immersion at 22-25° for 2-5 min followed by longer immersion in 30° water. Shulman (1960) suggested ice water treatment at 5-13° from 30 min to 5 h, depending on the severity of the injury. Wilson et al. (1963) found water at 16° for 15 min to 1 h to be most useful in the treatment of hot (63°) water burns. Despite these variations, it cannot be denied that cold water treatment of all types of thermal injury provides a quick and readily available therapy. However, what if the cold water cannot be applied immediately? King and Zimmerman (1965) have shown that 80-85% of the oedema which follows burn injury developed in the first 30 min although small amounts continued to form until the end of the third hour, but Harkins (1942, 1944) claims " it only completely subsides after 48 hours ". Thus, if cold is not applied immediately some oedema will still result. This is where the administration of the benzopyrones can be very important. The protein which leaks through the injured endothelium and enters the tissues (where it attracts oedema fluid) can be removed by proteolysis. This allows the small fragments to leave the tissues via the closed junctions of the blood vessels (Casley-Smith and Piller, 1974a,b; Piller, 1974a). The removal of protein frees the oedema fluid, with a resultant rapid resolution of the oedema. Since both cold and benzopyrone therapy are very effective in reducing burn oedema when given alone, the question was raised would they be better if given in combination? To date, there is no literature on the effectiveness of coumarin in the resolution of thermally induced oedema at temperatures other than those which must be assumed to be room temperature (F6ldiBorcsok et al., 1972; Casley-Smith et al., 1974; Casley-Smith and Piller, 1974a,b).
The amount of fluid loss into thermally injured regions, and thus the amount of oedema, is strongly dependent on blood flow and this on temperature (Courtice, 1946; Abramson, 1967). Thus, by keeping the animal cool, the outpouring of plasma into the injured regions can be reduced (Harkins, 1942, 1944; King and Zimmerman, 1965). Courtice (1946) reported a reduction in lymph flow by as much as 500% under cold treatment. The opposite has been observed as a consequence of the application of heat (Blalock and Baltimore, 1942). Thus, for the present experiments temperatures ranging from 10 to 350 with and without coumarin were used. Although it is well known that the lower range of temperatures used the lower tissue metabolic rate (Stone and Martin, 1958), it has no effect on the healing process of thermally injured limbs (Courtice, 1946). High temperatures were also tested although these were expected to be deleterious in aiding the resolution of thermal oedema (Blalock and Baltimore, 1942). The highest temperature of 350 was used in the light of a paper by Grayson (1949), in which it was shown temperatures of 36-40' caused the vasoconstriction of cutaneous vessels after their initial dilatation. It was thought that the vasodilatatory phase when combined with coumarin therapy might initially allow more rapid entry of the drug to the thermally injured regions, while the phase associated with vasoconstriction could possibly reduce the outpouring of protein and fluid from these vessels into the tissues. The net result might be a much more rapid rate of resolution. MATERIALS AND METHODS FoI the experiment 80 female albino rats, weight 200 ± 10 g, were used. Trwo days before the experiment both rear hind legs of all rats were closely shaven with an electric razor. They were then divided into 2 groups, each of which was then divided into 4 subgroups, 2 of which received intraperitoneal coumarin (25 mg/kg body weight in a 2% solution of A. R. ethyl alcohol in distilled water) immediately before the administration of bturn injtury. The other 2
85
TREATMENT OF THERMAL OEDEMA WITH COUMARIN
thermally induced oedema, they were of no use when this type of oedema was treated with coumarin at various ambient temperatures. Thus, a much simpler model was used. This consisted of 2 regression lines: one extending from zero time to the maximal recorded volumes and the other from this volume to the end of the observations. In this calculation, the values for each animal of each group were considered.
subgroups were given an equivalent volume of 2% A. R. ethyl alcohol in distilled water. All members of all subgroups were then given ether anaesthesia. Following this, the right hind limbs of all members of one group were burnt. For this the limbs were immersed in hot flowing water of 540 for 60 s. Temperature was maintained to within ± 0 5°. The injured limbs were then gently blotted dry with soft towelling. Following recovery from anaesthesia (5 min, at which stage there was no observable swelling on the injured limbs) the burnt rats with or without the coumarin therapy, were placed in wire cages in forced draught ventilated temperature cabinets, either at 10, 20, 30 or 35°. Adequate food and water were supplied. Simultaneously, all members of the other group were distributed to the various temperature cabinets. No members of this group were burnt. Two of the subgroups received coumarin while the others acted as a control, as before. Thus, at each temperature level there were 4 subgroups, viz., burn, burn with coumarin, normal (unburnt-untreated) and normal with coumarin. Measurements of both hind paws to the ankle joints were then performed by plethysmographic methods as described previously (Piller, 1974b). The times of measurement were immediately preceding thermal injury or entry into the temperature cabinets and at 3, 6, 7, 9, 10, 12, 16, 24, 48, 72, 130 and 200 h following injury or at the equivalent times for those groups without thermal injury. For each animal, corrections were made to the leg volumes to allow for the effect of temperature alone, both on the injured legs and on the normal ones. Oh-ishi and Sakuma (1970a, b) have suggested a number of simple models for rat paw oedema. Although these were claimed to fit
RESULTS
To ascertain the effectiveness of temperature with and without coumarin on thermally induced oedema, 5 parameters were examined. These were (1) the rate of swelling of the oedematous leg, (2) the maximal volume attained by the leg during the period of observation, (3) the time at which this maximal swelling was reached, (4) the rate of resolution of the oedema from the time of maximal swelling and (5) the time taken for the injured leg to return to normal.
1. Rate of swelling (Table I) In all cases from 10-35° the treatment of the oedema with coumarin increased the rate of swelling of the injured limb. However, the increase was only significant at 200 (0 001 < P < 0 01) and 350 (0 01< P < 0.05).
TABLE I.-Effect of Varying Temperatures ± Coumarin on the Rate of Swelling of Thermally Injured Limbs. Temp. 100 100 200 200
Treatment
Burn' Burn + coumarin2 Burn Burn + coumarin Burn Burn + coumarin
No. 5 5 5 5 5 5 5 5
Rate of swelling (ml/h) (X 10-2) 2-8
4.9
S.E. (X 10-3)
p3
8.3 10-2 8-6 12-0 5-3 7 9 10-5 10-0
4-3 ft 7-3 300 1-8 300 2-5 350 Burn 1-8 t 350 Burn + coumarin 5-8 1 Burn given by immersion of leg in hot (540), moving water for 1 min. 2 Coumarin, freshly prepared was administered i.p. at 25 mg/kg body weight in a 2 % solution of A.R. ethyl alcohol in physiological saline. Control groups received same volume of a 2 % solution of A.R. ethyl alcohol in saline. 3 Arrows
indicate direction and significance of change in oedema swelling -P>0 05; t 0 01
