<% vol = 14 number = 2 prevlink = 85 nextlink = 94 titolo = "ROLE OF LEUKOTRIENES IN THE PHYSIOPATHOLOGY OF THE RESPONSE TO EXPERIMENTAL BURN" volromano = "XIV" data_pubblicazione = "june 2001" header titolo %>

Santos X., Castilla C., Martín M., Arroyo C., Navarro L., Deiros L.

Experimental Surgery Unit, Hospital Universitario del Aire, Madrid, Spain

SUMMARY. The present study investigates how inhibition of the leukotrienes modifies the acute physiopathological response of the burned organism in the hypovolaemic shock phase, characterized by significant haemoconcentration. We studied the variations in haemoglobin and haematocrit both in the normal course of the general disease and in that in which the leukotrienes have been inhibited. Wistar rats were used to compare the possible effects of the leukotriene inhibitor ICI-198,615. The rats were divided into two groups (control and anti-leukotriene), treated with saline solution and the drug respectively in the first minutes after receiving a scald burn. The data were obtained by taking three blood samples from each animal (baseline, 15 min, and 60 min) in order to measure the variations in haemoglobin and haematocrit. Statistically significant differences between the two groups were observed in the average haematocrit and haemoglobin increases 15 min post-burn (p <0.05). These results prove the participation of leukotrienes in the extravasation that takes place during the first stages of the general disease in burn patients. We also deduce that the drug has an early maximum activity as its effects are apparent in the first 15 min post-burn, but not at later intervals.


Leukotrienes are active metabolites derived from arachidonic acid. This acid can be found in ester form in the phospholipids of the cell membrane; it is released by the action of phospholipase A2. Two enzymes (cyclo-oxygenase and lipo-oxygenase) act on arachidonic acid, and their action results respectively in the synthesis of prostaglandins and leukotrienes. Leukotrienes intervene in inflammatory processes, causing bronchoconstriction,1 an increase in vascular permeability,2 and vasoconstriction.3,4

Various substances (prostaglandins, leukotrienes, histamine, oxygen free radicals) are released after a burn. These lead to increased vascular permeability in the microcirculation5 and to plasma extravasation,6 resulting in hypovolaemia and haemoconcentration.7 It is known that thermal burns cause an activation of leukocytes that synthesize and release leukotrienes in the damaged tissue.8 It would therefore be interesting to study the effect of their inhibition or antagonism. Several studies on the pharmacological mechanisms of certain leukotriene antagonists have already been carried out.9,10 The purpose of our study was to investigate the action of one of these antagonists, ICI-198,615, on burns.

Leukotriene inhibitor ICI-198,615 is a potent and selective leukotriene receptor antagonist, reducing its effects on the microcirculation. Snyder et al.11 described its in vitro pharmacology, and Krell et al.12 described its in vivo pharmacology. Some studies showed its potent antagonistic effect on the actions of leukotrienes in asthma.13

Other studies showed the beneficial effect that the inhibition of leukotrienes had on the pathogenesis of asthma.1 Given that asthma is an inflammatory process, our study will attempt to determine the role of leukotrienes in the inflammatory response to burn.14

Material and methods

Twenty male Wistar rats (weight, 250-350 g) were anaesthetized intraperitoneally using a solution containing ketamine (50 mg/ml) at a dose of 75 mg/100 kg and medethomidine (1 mg/ml) at a dose of 0.5 mg/kg. Additional doses were given if necessary. We shaved the animal’s back from the neck to the base of the tail. A heparinized catheter (Venocath ® 18 g) 10 cm long was placed in the vena cava by dissecting the superficial femoral vein. The experimental burn model used in this study was scalding. The possibilities of changing the water temperature and of varying the exposure time and the area burned make this the ideal method for reproducing a thermal lesion.15 The rats were placed in special immobilization cages and were subsequently burned on 30% of the body surface area by immersion of the back of the animal in 70 °C water for 12 sec.

Two groups were set up (N° 10 rats in each). The first group was given an intravenous saline solution containing ICI-198,615 (concentration 1 mg/ml) at a dose of 0.125 ml/100 g immediately after burning. The second group (control group) was given an intravenous saline solution (0.125 ml/100 g) immediately after burning, and both groups were given 1 ml of saline solution intraperitoneally to control the immediate massive shock.14 Blood samples (0.3 ml) were taken before the burn, 15 min post-burn, and 60 min post-burn, in order to analyse the levels of haemoglobin (g/dl) and haematocrit (%).

During the study, care was taken at all times to comply with all government rules and regulations concerning animal experimentation, including those issued by the Council of the European Communities on Directive 86/609 (24 November 1986).

Statistical analysis: A pilot study was carried out using 10 rats, randomized into two groups of 5 rats each, treated respectively with the anti-leukotriene solution and saline solution. The data obtained were used to make a preliminary estimate of the sample size, aiming for an error <0.05 and an error < 0.2. The most important condition was the comparison of the levels of haematocrit and haemoglobin after 15 min, showing that a number of 10 rats per group was necessary.

Once the results of all 20 rats were obtained, we observed that the quantitative variables values did not deviate from the Gauss curve (Kolmogorov-Smirnoff). The quantitative variables were compared with Student’s t test. Owing to the diversity in baseline levels of haemoglobin and haematocrit between the different animals, a statistical analysis of the following increases in haemoglobin and haematocrit was used to homogenize the data of both groups: baseline-15', 15'-60', baseline-60'.


The data obtained indicated that in the control group the average increase in haemoglobin was -0.99 in the baseline-15' interval, -0.05 in the 15'-60' interval, and -1.04 in the baseline-60' interval; the values in the anti-leukotriene group were respectively +3.42, -1.93, and +1.49 (Fig. 1).

<% immagine "Fig. 1","90_fig_01.gif","Haemoglobin increments in control and anti-leukotriene groups.", 350 %>

The average haematocrit increases in the control group were -1.98 in the baseline-15' interval, -1.05 in the 15'-60' interval, and -3.03 in the baseline-60' interval; the values in the anti-leukotriene group were respectively +9.38, -34.74, and -25.3 (Fig. 2).

<% immagine "Fig. 2","90_fig_02.gif","Haematocrit increments in control and anti-leukotriene groups.", 350 %>

The statistical data showed a statistically significant difference in the haemoglobin levels of both groups for the baseline-15' interval (p = 0.027) and the 15'-60' interval (p = 0.037). There was also a statistically significant difference in the haematocrit levels of both groups for the baseline-15' interval (p = 0. 035).


Our data suggest that the administration of ICI-198,615 in rats immediately after a thermal lesion may contribute to the partial reversibility of the effects derived from the action of leukotrienes. Nicosia et al.13 have shown the effects of the drug as a leukotriene antagonist in bronchial constriction and the increased vascular permeability that these metabolites cause in asthma. Our results showed a lower degree of haemoconcentration in the anti-leukotriene group than was seen in the control group, as indicated by a lower increase in haemoglobin and haematocrit in the treated animals. Considering the different intervals analysed, we noted that there was a statistically significant lower haemoconcentration during the first 15 min post-burn. Even though the half-life of the drug administered i.v. in guinea-pigs has been shown to be 68 min,12 we found that its activity was only maintained up to the time of taking the first blood sample (baseline-15'), and that there were no significant differences after 60' either in haemoglobin or in haematocrit, with the exception of haemoglobin for the 15'-60' interval. Taking into consideration that the haematocrit is the main parameter for haemoconcentration, we deduce that the pharmacological action was predominant during the immediate acute post-burn phase.

It is known that in thermal burns there is an activation of leukocytes that synthesize and release leukotrienes to the tissue.8 Four different indications have implicated these arachidonic acid metabolites as mediators for ischaemia and shock: a) the increased level of extravasal liquid during the shock phase; b) the capacity to exert significant physiopathological effects that increase ischaemia in this entity; c) the reduced degree of shock with leukotriene synthesis inhibitors and antagonists to their receptors; and d) the induction of a state of shock after the exogenous administration of leukotrienes.16 Other studies have shown that in the initial post-burn period there is a release of arachidonic acid metabolites, particularly of leukotriene B4.17 This metabolite is considered to be the most powerful modulator of the polymorphonuclear cells, the essential cells in the earliest stages of inflammation, stimulating lysosomal degranulation, aggregation, and adherence.18

Levitt et al.19 used CGP 33304 and CGP 35949 respectively as receptor antagonist and phospholipase A2 inhibitor, thus avoiding the hypoxia that is secondary to the bronchial constriction induced by leukotrienes. The drug ICI 198,615 is a potent leukotriene receptor antagonist. Guinea-pigs given LTD4 in spray form showed how the effects of the leukotrienes were reversed when this drug was administered. The pharmacological half-life when administered orally, intravenously, and by inhalation was respectively > 16 h, 68 min, and 34 min.12

Considering the leukotriene neutralizing action, the administration of ICI-198,615 could partially reverse the physiopathological alterations caused by the burn. The haemoglobin and haematocrit levels increased less in our anti-leukotriene group than in our control group. The difference between these levels was greatest in the baseline-15' post-burn period for both haemoglobin and haematocrit (p <0.05), and there was also a statistically significant difference in the 15'-60' interval for the haemoglobin levels (p < 0.05). These results reflect the fact that the levels of haemoglobin and haematocrit did not increase with respect to the baseline levels, which did however occur in the control group, and this is probably due to the extravasation inhibitory action of the ICI-198,615 drug as a potent antagonist of the leukotriene receptors. The fact that the differences in the haematocrit and haemoglobin levels between the two groups at 60 min post-burn is not significant is probably due to the short duration of the effect in rats. We presume that the administration of a new dose of the drug in the 15'-60' post-burn interval, or by continuous infusion, would yield statistically significant differences in the values of both groups. This will be the objective of a future study.

In conclusion, our data suggest that the phenomenon of haemoconcentration that occurs in the general disease in burn patients is at least partially related to the leukotriene levels, and that the administration of ICI-198,615 after a burn may decrease these processes to a greater or lesser degree. An exhaustive bibliographic search has not yielded any studies that associate the effects of inhibiting leukotrienes and burns.

RESUME. Les Auteurs ont étudié comment l’inhibition des leucotriènes modifie la réponse physiopathologique aiguë de l’organisme brûlé pendant la phase du choc hypovolémique, caractérisée par une hémoconcentration significative. Ils ont considéré les variations de l’hémoglobine et de l’hématocrite soit pendant le cours normal de la maladie générale soit dans le cas où les leucotriènes ont subi un effet inhibiteur. Des rats Wistar ont été employés pour comparer les effets possibles de l’inhibiteur des leucotriènes ICI-198,615. Les rats ont été divisés en deux groupes (groupe témoin et groupe anti-leucotriènes), traités respectivement avec de la solution saline et la substance chimique dans les premiers minutes après la brûlure par l’ébouillantement. Les résultats ont été obtenus moyennant trois prélèvements de sang de chaque animal (ligne de base, 15 minutes et 60 minutes) pour mesurer la variation de l’hémoglobine et de l’hématocrite. Les Auteurs ont observé des différences statitistiquement significatives entre les deux groupes pour ce qui concerne les augmentations moyennes de l’hématocrite et de l’hémoglobine 15 minutes après la brûlure (p <0,05). Les résultats démontrent la participation des leucotriènes dans l’extravasation qui si vérifie pendant les premières phases de la maladie générale dans les patients brûlés. Les Auteurs concluent que cette substance effectue une activité maximale précoce, puisque ses effets sont évidents dans les premiers 15 minutes après la brûlure, mais non par la suite.


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<% riquadro "This paper was received on 1 March 2001.

Address correspondence to:
Dr X. Santos, Ph.D., Experimental Surgery Unit, Hospital Universitario del Aire,
Arturo Soria 82, 28027, Madrid, Spain."%>

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