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Evaluation of a leukotriene receptor antagonist in prevention of hyperoxic lung injury in newborn rabbits
Rostaglmdins Leukotriener and Essential Fatty Acids (19%) 0 hmam Group UK Ltd 1992
45. 15%I65
Evaluation of a Leukotriene Receptor Antagonist in Prevention of Hyperoxic Lung Injury in Newborn Rabbits N. J. Kertesz, R. B. Holtzman,
L. Adler and J. R. Hageman
The Department of Pediatrics, Evanston Hospital, 2650 Ridge Avenue, Northwestern University Medical School, Evanston, Illinois 60201, USA (Reprint requests to RBH) ABSTRACT. Prolonged exposure to hyperoxia can result in sign&ant hmg @jury and has been associated with the development of bronchopulmonary dysplasia. Leukotrienes (LT) recruit polymo@onuclear leukocytes (PMN) to the lung, increase vascular permeability, and have therefore been postulated to play a roie in the pa&genesis of hyperoxic lung injury. This study investigates ICI 198,615 (ICI), an LTD4 and LTE4 receptor antagonist in preventing hyperoxic lung injury in newborn rabbits. Matched littermates of %day-oM rabbii received ICI (0.1 or 1.0 j.&@g/h) or vehicle aione, were exposed to >95% 02, and sacriBced after 48,72,84 and 96 h of exposure. Bronchoalveolar lavage iiuid (BAL) of the left lung was analyzed for white cell count, differential, absoh& number of PMNs, total protein, and cyclooxygenase products Qketo-PGF1,, and thromboxane Bz. Lung water was quantified utilizing the right lung. Results demonstrated no significant differences between the ICI groups or between the ICI groups and controls. In conchwion, the administration of the LTD4 and LTEd receptor antagonist ICI 19%,615 was in&Went to reduce the formation of pulmonary edema, reduce mortality or attenuate hyperoxic hmg injury. These experiments suggest that a number of other mediators may be involved in the hyperoxic lung injury process and that the functional inhibition of a portion of the arachidonic acid cascade was not suflicient to either prevent or attenuate hyperoxic lung injury in newborn rabbits.
INTRODUCTION
tulated to act as second messengers that amplify the injury caused by oxygen radicals. An in vivo experiment performed in mice showed that LTD4 was found in the bronchoalveolar lavage (BAL) fluid of mice exposed to 100% oxygen for 2 days. LTD., continued to increase with longer exposure and then decreased while LTEd appeared when lung damage became severe (14). LTD4 has been shown to cause pulmonary hypertension in guinea-pigs, sheep and monkeys (15-17). LTs have also been shown to be important mediators in the generation of pulmonary edema (11, 18, 19). In in vitro experiments pulmonary hypertension and pulmonary edema are proposed to be manifestations of oxidant induced pulmonary toxicity (9, 11, 20, 21). Age also seems to play a role in an animal’s ability to detoxify oxygen radicals. Our previous work found that l- to %-day-old newborn rabbits were tolerant to 65 h of hyperoxic exposure. In contrast, an equal duration of exposure in ‘jr-day-old rabbits resulted in significant lung injury. We have also shown that lo-day-old rabbits exposed to ~95% oxygen had evidence of microscopic lung injury and
High concentrations of inspired oxygen are often necessary in the treatment of critically ill infants and adults. However, prolonged exposure to hyperoxia has been associated with serious pulmonary injury. There is evidence indicating that oxygen metabelites play an important pathophysiological role in many types of lung inflammation and injury (l-6). It has been postulated that oxygen toxicity begins with the production of oxygen radicals. Oxygen metabolites have been shown to activate phospholipase A2 and cause the release and metabolism of arachidonic acid in isolated perfused lungs (7, 8). Arachidonic acid metabolites have the capacity to modulate the inflammatory response and have been implicated as mediators of a broad range of processes in the lung (9-13). These metabolites, specifically the leukotrienes (LT), have been pos-
Date received 6 September 1991 Date accepted 30 September 1991 159
160
Prostaelandins
Leukotrienes
and Essential Fattv Acids
demonstrated increases in BAL white cells, neutrophils, total protein, TXBz, and 6-keto-PGFr, (22). We performed the following in vivo experiments in order to examine whether the administration of an LTD4 and LTE4 receptor antagonist (ICI) will protect against the development of hyperoxic lung injury and thereby decrease morbidity and mortality secondary to the injury process. This antagonist, ICI 198,615 (a generous gift from Dr Robert Krell of Stuart Pharmaceuticals) has been reported to be one of the most potent antagonists of LTD4 and LTE1, (23).
MATERIALS
AND METHODS
Litters of full term, 7-day-old, Pasteurella-free, New Zealand albino rabbits and their dam were obtained from Lesser’s Rabbitry (Union Grove, WI). Litters varied in size from 4 to 16, each of which was divided into pairs based upon weight. The ICI experimental group was randomly assigned one rabbit from each pair and the other served as its control. Each rabbit’s back was cleaned, anesthetized with 1% lidocaine, and a 1 cm skin incision was made. A micro-pump (Alza Corp., Palo Alto, CA) dispensing at a rate of 0.5 uI_& was placed subcutaneously in each rabbit. ICI was dissolved in a vehicle consisting of polyethylene glycol 400 (PEG 4OO), 1 M NaOH, and phosphate buffered saline (PBS). ICI doses were chosen based on a paper by Krell et al outlining the in vivo pharmacology of the drug (24). In acute experiments on guinea-pigs measuring ICI’s antagonism of LTD4 induced dyspnea, it was found that a dose of 0.1 uM/kg was sufficient to maximally antagonize the effects of LTD4 (24). To investigate the possibility of a dose related phenomenon, we also used a dose of 1.0 t&I/k&r. The ICI rabbits received the antagonist at a dose of 0.1 or 1.0 t&I/kg/h, and the control rabbits received a pump containing the vehicle alone. The wound was closed with 2-3 stitches of nylon suture. The animals were allowed 4 h of recovery, the time needed for the pumps to begin to function, before being placed in >95% oxygen. Hyperoxic conditions were maintained in sealed polyethylene cages (Nalge, Rochester, NY) and soda lime (W. R. Grace, Lexington, MA) was used to remove carbon dioxide. FiOz was monitored polygraphically (Instrumentation Laboratories, Lexington, MA). Nursing newborns were allowed access to their dam for 16 out of each 24 h under hyperoxic conditions. The dam was removed to room air for 8 daytime h per day. Litters, receiving ICI at a dose of 0.1 &I/kg/h, were studied following 48 (N = 4), 72 (N = l), 84 (N = 3), and 96 (N = 6) h of oxygen exposure and those receiving ICI at
1.0 t&I/kg/h were studied after 84 (N = 8) and 96 (N = 9) hours of exposure. Control animals were studied after 48 (N = 2), 72 (N = 2), 84 (N = ll), and 96 (N = 12) h of exposure to >95% oxygen. Rabbits were anesthetized with 50 mg/kg pentobarbital injected intraperitoneally and sacrificed via aortic exsanguination. The diaphragm was pierced, and the rib cage was cut away to gain access to the lungs. A nylon tie was placed around the right mainstem bronchus, thereby occluding it, to prevent saline from entering it during the BAL of the left lung. The trachea was cannulated using a 16 gauge angiocath, and BAL was then performed on the left lung using 2 ml of 0.9% normal saline at room temperature. The lung was ravaged five times (total volume = 10 ml) and the effluent was combined and placed on ice. Yields from the BAL were consistently >90% of the 2 ml aliquots. The effluent was then centrifuged at 1000 g for 10 min at 4°C. Aliquots of the supernatant were stored at -70°C for analysis of BAL protein and arachidonic acid metabolites. The cell pellet was resuspended in saline and counted on a hemocytometer. Cytospin preparations of BAL cells were stained with Wright-Giemsa for white cell differential counts. Following this procedure, the right lung was removed, trimmed of all non-parenchymal tissue, blotted and weighed. It was then placed in a dry heat incubator and reweighed until the dry weight had stabilized. Lung water was measured by using lung wet weight to body weight ratios and lung wet to dry ratios. Protein content of BAL fluid was measured by the Coomasie blue dye method (Bio-Rad, Richmond, CA) using fresh bovine serum albumin (Sigma, St. Louis, MO) as a reference standard. Cyclooxygenase metabolites 6-keto-PGF1, (the stable metabolite of PG12) and TXB2 (the metabolite of TXA*) were measured from BAL samples using standard radioimmunoassay (RIA) methods as previously published (25). Eicosanoid standards were obtained from Sigma. 100 microliter sample volumes were incubated with 100 ~1 of tritiated tracer (Amersham, Arlington Heights, IL) and 100 PL of antibody (Advanced Magnetics, Boston, MA) at 4°C for 16 h. 200 PL of dextran-coated charcoal was then added, and 0.4 mL of the supernatant was decanted for scintillation counting. Inter-assay variability was monitored by spiking random samples with known quantities of standard. The recovery based on this technique was determined to be >90%. A second experiment was also performed to determine mean survival in >95% oxygen. The rabbits were prepared in the same manner as outlined above utilizing ICI at a dose of ICI 0.1 @I/kg/h vs vehicle. The rabbits were placed ‘in hyperoxic conditions and checked every 10-12 h. Data were analyzed using an unpaired two-tailed
LT Receptor Antagonist and Prevention of Hyperoxic Lung Injury
Student’s t-test. A p value of <.05 was considered significant.
NEUTROPHIL X
161
INFLUX
PMN
35
30
i
RESULTS Minimal differences in biologic parameters of lung injury between the ICI and control groups were noted following 48 and 72 h of exposure to hyperoxia. Therefore the majority of animal subjects were utilized studies of longer duration. Figure 1 summarizes the amount of total protein found in BAL fluid as a function of the length of hyperoxic exposure. Hyperoxic exposure for 48 and 72 h resulted in low levels of BAL total protein in both control and ICI groups. However, as exposure was increased to 84 and 96 h BAL levels of total protein increased in all groups. However, no significant differences were found between either the ICI groups and the control group. TOTAL PROTEIN Proteh 600
(N/ml
1
500
I
C AIx#PPMNLxl05)
41 3j elOi-
I
40
,
72
-
Control
----ICI
IO
IpM/kg/hr)
---ICI
(Ol~~M/kg/hr)
.‘.’ ..”
“ICI
(l.O~~M/kg/hr)
T
LUNG
Control
96
Fig. 2 A: PMNS, represented as a percentage of the total white cells recovered from BAL of the left lung, vs hours of exposure to >9.5% oxygen. B. PMNs, represented as the absolute number recovered from BAL of the left lung, x lo’ vs hours of exposure to >95% oxygen. The bars represent standard errors of the mean.
LUNG
-
1
84 Howt of Exposure
WET
DRY
EDEMA
RATIOS
T
ICI(I.O~M/kg/hr)
Fig. 1 Total protein recovered from BAL of the left lung in ug/ml vs hours of exposure to >95% oxygen. The bars represent standard errors of the mean.
The percentage and absolute numbers of PMNs recovered from BAL, were very low at 48 and 72 h as presented in Figures 2A and B. Increases in both were noted with more prolonged hyperoxic exposure, a pattern very similar to what was observed with total protein. However, no statistically significant differences were found between either ICI group and the control group at each of the time intervals studied for either of these measurements. Figures 3A and B summarize the accumulation of lung water after hyperoxic exposure. Lung wet weight: body weight ratios began to increase at 72 h in all three groups and continued to increase slowly after 84 and 96 h of hyperoxic exposure. Again, no differences were found between the ICI and the control group in lung wet weight to body weight or lung wet to dry ratios.
LUNG WET
WT/BODY
WT
04--
1
48
72
84 Hours
-
control
---
96
of Expowre
ICI (0 I rrM/kq/hr)
“’ ICI
ll.O~/kg/hr)
Fig. 3 A: Lung water expressed as lung wet:dry ratios of the right lung vs hours of exposure to >95% oxygen. B: Lung water expressed as lung wet weight to body weight ratios vs hours of exposure to >95% oxygen. The bars represent standard errors of the mean.
162
Prostaglandins Leukotrienes
and Essential Fatty Acids
CYCLOOXYGENASE PRODUCTS IN BAL FLUID
I
4001
c .......... .......*......... ~;I”... Jr
300200-
DISCUSSION
IOO-
TX82 300200
significant difference between the ICI and the control group (n = 60) at the time of their death or at any stage of the study.
//M
-
. ..Yzm--W~.__~
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O!
72
48
f 96
84 tlours of Exposure
-
Control
---
ICI (0 I yM/kg/hr)
‘..
.. ICI (IOyM/kg/hr)
Fig. 4 A: 6-Keto-PGF,,, the stable metabolite of PGI,, in pg/ml vs hours of exposure to >95% oxygen. B: TXB,, the stable metabolite of TXA,, in pg/ml vs hours of exposure to >95% oxygen. The bars represent standard errors of the mean.
Figures 4A and B summarize BAL fluid levels of cyclooxygenase products 6-keto-PGFi, vs hours of hyperoxic exposure and TXB2 vs hours of hyperoxic exposure. They were, as were the total protein and polymorphonuclear cell influx, relatively low after 48 and 72 h of oxygen exposure. After 84 and 96 h of hyperoxia both of these products increased in BAL fluid. Again, there were no significant differences between the three groups at any stage of the study. Figure 5 shows the mortality rates in relation to the duration of exposure to hyperoxia of ICI and control animals. 50% mortality was found to be at 72 h of hyperoxic exposure. There were no significant differences at any time between the ICI and the control group. The rabbits were also weighed daily to determine if their mother was showing preference for either the ICI or control animals and to see if the drug itself was affecting their development. There was no
MORTALITY % mortality 1001 80’
84
60 Hours -
Control
---
ICI (0
I
108
7
132
of Exposure
I rrU/kg/hr)
Fii. 5 The percent mortality of the rabbits at any given number of hours of exposure to >95% oxygen (ICI) vs hours of exposure to >95% oxygen (control).
Our data demonstrate that the administration of a specific leukotriene D4 and E4 receptor antagonist to oxygen susceptible, maturing, 7-day-old rabbits does not have a protective effect against the development of oxygen induced lung injury. Specifically, the administration of this compound did not inhibit PMN influx, the development of proteinaceous pulmonary edema, or improve survival with prolonged oxygen exposure. Several possible mechanisms may have contributed to these results: 1. Oxygen radicals by themselves are very toxic to the lung and their unopposed actions could play a significant and primary role in hyperoxic lung injury. 2. Other biochemical and cellular mediators, e.g. LTB4, neutrophils, macrophages, and lipoxins, may play a greater role than previously appreciated in in vitro experiments. 3. The drug did not inhibit the actions of LTD,+. The free radical theory of oxygen toxicity has generally been accepted as the biochemical and molecular mechanism that best explains injury secondary to hyperoxia (26). Oxygen radicals alone may cause sufficient injury to induce mortality by inactivating enzymes, peroxidizing membrane double bonds, causing alterations in membranes, cross-linking and causing schisms of DNA fragments, and causing sulfhydryl oxidation of protein moieties (26). The superoxide radical itself has been shown to damage and kill cells (27-30). Oxygen induced injury to endothelial and epithelial barriers of alveolar walls can cause pulmonary edema (26). All these effects, by themselves or in combination, may be sufficient to cause extensive injury and even mortality. The preceding paragraph may in part explain why the administration of a LTD4 and LTE4 antagonist did not inhibit the development of pulmonary edema. A difficulty in examining oxygen induced lung injury is that phenomena that occur in vitro may not be important or may not occur in vivo. Lipoxygenase products have been shown to increase systemic vascular permeability (31-33) and appear to increase pulmonary vascular permeability in a number of species including the rabbit (9, 11, 19, 34-36). This was further supported when Farrukh et al (19) showed in an in vitro experiment that the administration of LTD4 caused an increase in pulmonary vascular permeability in rabbits. While LTD4 may be one of the main mediators of pul-
LT Receptor Antagonist and Prevention of Hyperoxic Lung Injury
monary edema and oxidant induced pulmonary injury in vitro, there may be many other cellular and biochemical mediators at work in the intact animal that may not be evident in vitro. While the administration of ICI may have completely blocked the effects of LTD4, other unopposed mediators may be sufficient to produce pulmonary edema and lung injury. An important cell and source of mediators that may not be evident in in vitro experiments is the PMN. Polymorphonuclear leukocytes may play an important role in regulating fluid efflux from venules. This may imply that potent chemoattractant leukotrienes, e.g. LTB4, may also indirectly increase tissue edema through the recruitment of neutrophils (37). While ICI 198,615 is a leukotriene receptor antagonist with a high affinity for LTD4 and LTE4 (38), it has no effect against LTB4. LTB4 is one of the most potent chemokinetic and chemotactic agents known, able to affect the human, rat and rabbit PMN. It has therefore been proposed that LTB4 has a role in leukocyte recruitment during inflammation (31, 39, 40). The accumulation of neutrophils has been further postulated to contribute to lung injury during hyperoxia (37). It was also found that the exposure of rats to hyperoxia caused a marked increase in the chemoattractant activity of BAL fluid that was correlated with the increase in the number of PMNs in lung ravages. These events were followed by the death of the animal in a few hours (41). White cells, including macrophages, also actively participate in the further generation of active oxygen species (42), e.g. the superoxide anion, hydrogen peroxide and the hydroxyl radical (43-45), which can damage and kill cells. Leukotoxins, metabolites of the 1Slipoxygenase pathway, may also contribute to oxidant induced lung injury. Phospholipase A2 has been shown to cause the release of these products in addition to the leukotrienes via the release of the precursors of arachidonic acid. These products have been isolated from lung lavages of rats breathing pure oxygen and have been shown to have an uncoupling effect of mitochondrial respiration (46). Cyclooxygenase products in the current study were not increased by the ICI-mediated inhibition of LT function. The fact that 50% mortality occurred at 72 h in our studies was somewhat surprising. At that point i.n time only the lung wet weight to body weight ratios had begun to increase. All other indicators of injury in the lung, e.g. total protein, cyclooxygenase products, and neutrophil influx (Figs 1, 2 & S), had not shown any change from the 48-h measurement. This can be interpreted to mean that pulmonary edema in the absence of any other injury may be sufficient to cause mortality. It may also mean that the severe direct toxic
163
effects of oxygen radicals and neutrophils to the cells themselves were also enough to result in mortality. Oxygen may also be toxic to other organ systems so that the time of mortality is not based solely on sufficient damage to the lungs. In conclusion, the administration of a leukotriene D4 and E$ receptor antagonist was insufficient to stop the formation of pulmonary edema, attenuate mortality, or attenuate pulmonary injury secondary to hyperoxia. This experiment suggests that a number of other mediators may be involved in lung injury and that the inhibition of only one arm of the arachidonic acid cascade may not be sufficient to either prevent or attenuate oxygen induced injury. Acknowledgements Supported in part by the Dee and Moody Research Fund of Evanston Hospital. Presented in part at the Society for Pediatric Research, May 1989.
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Leukotrienes
and Essential Fattv Acids
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45. 15%I65
Evaluation of a Leukotriene Receptor Antagonist in Prevention of Hyperoxic Lung Injury in Newborn Rabbits N. J. Kertesz, R. B. Holtzman,
L. Adler and J. R. Hageman
The Department of Pediatrics, Evanston Hospital, 2650 Ridge Avenue, Northwestern University Medical School, Evanston, Illinois 60201, USA (Reprint requests to RBH) ABSTRACT. Prolonged exposure to hyperoxia can result in sign&ant hmg @jury and has been associated with the development of bronchopulmonary dysplasia. Leukotrienes (LT) recruit polymo@onuclear leukocytes (PMN) to the lung, increase vascular permeability, and have therefore been postulated to play a roie in the pa&genesis of hyperoxic lung injury. This study investigates ICI 198,615 (ICI), an LTD4 and LTE4 receptor antagonist in preventing hyperoxic lung injury in newborn rabbits. Matched littermates of %day-oM rabbii received ICI (0.1 or 1.0 j.&@g/h) or vehicle aione, were exposed to >95% 02, and sacriBced after 48,72,84 and 96 h of exposure. Bronchoalveolar lavage iiuid (BAL) of the left lung was analyzed for white cell count, differential, absoh& number of PMNs, total protein, and cyclooxygenase products Qketo-PGF1,, and thromboxane Bz. Lung water was quantified utilizing the right lung. Results demonstrated no significant differences between the ICI groups or between the ICI groups and controls. In conchwion, the administration of the LTD4 and LTEd receptor antagonist ICI 19%,615 was in&Went to reduce the formation of pulmonary edema, reduce mortality or attenuate hyperoxic hmg injury. These experiments suggest that a number of other mediators may be involved in the hyperoxic lung injury process and that the functional inhibition of a portion of the arachidonic acid cascade was not suflicient to either prevent or attenuate hyperoxic lung injury in newborn rabbits.
INTRODUCTION
tulated to act as second messengers that amplify the injury caused by oxygen radicals. An in vivo experiment performed in mice showed that LTD4 was found in the bronchoalveolar lavage (BAL) fluid of mice exposed to 100% oxygen for 2 days. LTD., continued to increase with longer exposure and then decreased while LTEd appeared when lung damage became severe (14). LTD4 has been shown to cause pulmonary hypertension in guinea-pigs, sheep and monkeys (15-17). LTs have also been shown to be important mediators in the generation of pulmonary edema (11, 18, 19). In in vitro experiments pulmonary hypertension and pulmonary edema are proposed to be manifestations of oxidant induced pulmonary toxicity (9, 11, 20, 21). Age also seems to play a role in an animal’s ability to detoxify oxygen radicals. Our previous work found that l- to %-day-old newborn rabbits were tolerant to 65 h of hyperoxic exposure. In contrast, an equal duration of exposure in ‘jr-day-old rabbits resulted in significant lung injury. We have also shown that lo-day-old rabbits exposed to ~95% oxygen had evidence of microscopic lung injury and
High concentrations of inspired oxygen are often necessary in the treatment of critically ill infants and adults. However, prolonged exposure to hyperoxia has been associated with serious pulmonary injury. There is evidence indicating that oxygen metabelites play an important pathophysiological role in many types of lung inflammation and injury (l-6). It has been postulated that oxygen toxicity begins with the production of oxygen radicals. Oxygen metabolites have been shown to activate phospholipase A2 and cause the release and metabolism of arachidonic acid in isolated perfused lungs (7, 8). Arachidonic acid metabolites have the capacity to modulate the inflammatory response and have been implicated as mediators of a broad range of processes in the lung (9-13). These metabolites, specifically the leukotrienes (LT), have been pos-
Date received 6 September 1991 Date accepted 30 September 1991 159
160
Prostaelandins
Leukotrienes
and Essential Fattv Acids
demonstrated increases in BAL white cells, neutrophils, total protein, TXBz, and 6-keto-PGFr, (22). We performed the following in vivo experiments in order to examine whether the administration of an LTD4 and LTE4 receptor antagonist (ICI) will protect against the development of hyperoxic lung injury and thereby decrease morbidity and mortality secondary to the injury process. This antagonist, ICI 198,615 (a generous gift from Dr Robert Krell of Stuart Pharmaceuticals) has been reported to be one of the most potent antagonists of LTD4 and LTE1, (23).
MATERIALS
AND METHODS
Litters of full term, 7-day-old, Pasteurella-free, New Zealand albino rabbits and their dam were obtained from Lesser’s Rabbitry (Union Grove, WI). Litters varied in size from 4 to 16, each of which was divided into pairs based upon weight. The ICI experimental group was randomly assigned one rabbit from each pair and the other served as its control. Each rabbit’s back was cleaned, anesthetized with 1% lidocaine, and a 1 cm skin incision was made. A micro-pump (Alza Corp., Palo Alto, CA) dispensing at a rate of 0.5 uI_& was placed subcutaneously in each rabbit. ICI was dissolved in a vehicle consisting of polyethylene glycol 400 (PEG 4OO), 1 M NaOH, and phosphate buffered saline (PBS). ICI doses were chosen based on a paper by Krell et al outlining the in vivo pharmacology of the drug (24). In acute experiments on guinea-pigs measuring ICI’s antagonism of LTD4 induced dyspnea, it was found that a dose of 0.1 uM/kg was sufficient to maximally antagonize the effects of LTD4 (24). To investigate the possibility of a dose related phenomenon, we also used a dose of 1.0 t&I/k&r. The ICI rabbits received the antagonist at a dose of 0.1 or 1.0 t&I/kg/h, and the control rabbits received a pump containing the vehicle alone. The wound was closed with 2-3 stitches of nylon suture. The animals were allowed 4 h of recovery, the time needed for the pumps to begin to function, before being placed in >95% oxygen. Hyperoxic conditions were maintained in sealed polyethylene cages (Nalge, Rochester, NY) and soda lime (W. R. Grace, Lexington, MA) was used to remove carbon dioxide. FiOz was monitored polygraphically (Instrumentation Laboratories, Lexington, MA). Nursing newborns were allowed access to their dam for 16 out of each 24 h under hyperoxic conditions. The dam was removed to room air for 8 daytime h per day. Litters, receiving ICI at a dose of 0.1 &I/kg/h, were studied following 48 (N = 4), 72 (N = l), 84 (N = 3), and 96 (N = 6) h of oxygen exposure and those receiving ICI at
1.0 t&I/kg/h were studied after 84 (N = 8) and 96 (N = 9) hours of exposure. Control animals were studied after 48 (N = 2), 72 (N = 2), 84 (N = ll), and 96 (N = 12) h of exposure to >95% oxygen. Rabbits were anesthetized with 50 mg/kg pentobarbital injected intraperitoneally and sacrificed via aortic exsanguination. The diaphragm was pierced, and the rib cage was cut away to gain access to the lungs. A nylon tie was placed around the right mainstem bronchus, thereby occluding it, to prevent saline from entering it during the BAL of the left lung. The trachea was cannulated using a 16 gauge angiocath, and BAL was then performed on the left lung using 2 ml of 0.9% normal saline at room temperature. The lung was ravaged five times (total volume = 10 ml) and the effluent was combined and placed on ice. Yields from the BAL were consistently >90% of the 2 ml aliquots. The effluent was then centrifuged at 1000 g for 10 min at 4°C. Aliquots of the supernatant were stored at -70°C for analysis of BAL protein and arachidonic acid metabolites. The cell pellet was resuspended in saline and counted on a hemocytometer. Cytospin preparations of BAL cells were stained with Wright-Giemsa for white cell differential counts. Following this procedure, the right lung was removed, trimmed of all non-parenchymal tissue, blotted and weighed. It was then placed in a dry heat incubator and reweighed until the dry weight had stabilized. Lung water was measured by using lung wet weight to body weight ratios and lung wet to dry ratios. Protein content of BAL fluid was measured by the Coomasie blue dye method (Bio-Rad, Richmond, CA) using fresh bovine serum albumin (Sigma, St. Louis, MO) as a reference standard. Cyclooxygenase metabolites 6-keto-PGF1, (the stable metabolite of PG12) and TXB2 (the metabolite of TXA*) were measured from BAL samples using standard radioimmunoassay (RIA) methods as previously published (25). Eicosanoid standards were obtained from Sigma. 100 microliter sample volumes were incubated with 100 ~1 of tritiated tracer (Amersham, Arlington Heights, IL) and 100 PL of antibody (Advanced Magnetics, Boston, MA) at 4°C for 16 h. 200 PL of dextran-coated charcoal was then added, and 0.4 mL of the supernatant was decanted for scintillation counting. Inter-assay variability was monitored by spiking random samples with known quantities of standard. The recovery based on this technique was determined to be >90%. A second experiment was also performed to determine mean survival in >95% oxygen. The rabbits were prepared in the same manner as outlined above utilizing ICI at a dose of ICI 0.1 @I/kg/h vs vehicle. The rabbits were placed ‘in hyperoxic conditions and checked every 10-12 h. Data were analyzed using an unpaired two-tailed
LT Receptor Antagonist and Prevention of Hyperoxic Lung Injury
Student’s t-test. A p value of <.05 was considered significant.
NEUTROPHIL X
161
INFLUX
PMN
35
30
i
RESULTS Minimal differences in biologic parameters of lung injury between the ICI and control groups were noted following 48 and 72 h of exposure to hyperoxia. Therefore the majority of animal subjects were utilized studies of longer duration. Figure 1 summarizes the amount of total protein found in BAL fluid as a function of the length of hyperoxic exposure. Hyperoxic exposure for 48 and 72 h resulted in low levels of BAL total protein in both control and ICI groups. However, as exposure was increased to 84 and 96 h BAL levels of total protein increased in all groups. However, no significant differences were found between either the ICI groups and the control group. TOTAL PROTEIN Proteh 600
(N/ml
1
500
I
C AIx#PPMNLxl05)
41 3j elOi-
I
40
,
72
-
Control
----ICI
IO
IpM/kg/hr)
---ICI
(Ol~~M/kg/hr)
.‘.’ ..”
“ICI
(l.O~~M/kg/hr)
T
LUNG
Control
96
Fig. 2 A: PMNS, represented as a percentage of the total white cells recovered from BAL of the left lung, vs hours of exposure to >9.5% oxygen. B. PMNs, represented as the absolute number recovered from BAL of the left lung, x lo’ vs hours of exposure to >95% oxygen. The bars represent standard errors of the mean.
LUNG
-
1
84 Howt of Exposure
WET
DRY
EDEMA
RATIOS
T
ICI(I.O~M/kg/hr)
Fig. 1 Total protein recovered from BAL of the left lung in ug/ml vs hours of exposure to >95% oxygen. The bars represent standard errors of the mean.
The percentage and absolute numbers of PMNs recovered from BAL, were very low at 48 and 72 h as presented in Figures 2A and B. Increases in both were noted with more prolonged hyperoxic exposure, a pattern very similar to what was observed with total protein. However, no statistically significant differences were found between either ICI group and the control group at each of the time intervals studied for either of these measurements. Figures 3A and B summarize the accumulation of lung water after hyperoxic exposure. Lung wet weight: body weight ratios began to increase at 72 h in all three groups and continued to increase slowly after 84 and 96 h of hyperoxic exposure. Again, no differences were found between the ICI and the control group in lung wet weight to body weight or lung wet to dry ratios.
LUNG WET
WT/BODY
WT
04--
1
48
72
84 Hours
-
control
---
96
of Expowre
ICI (0 I rrM/kq/hr)
“’ ICI
ll.O~/kg/hr)
Fig. 3 A: Lung water expressed as lung wet:dry ratios of the right lung vs hours of exposure to >95% oxygen. B: Lung water expressed as lung wet weight to body weight ratios vs hours of exposure to >95% oxygen. The bars represent standard errors of the mean.
162
Prostaglandins Leukotrienes
and Essential Fatty Acids
CYCLOOXYGENASE PRODUCTS IN BAL FLUID
I
4001
c .......... .......*......... ~;I”... Jr
300200-
DISCUSSION
IOO-
TX82 300200
significant difference between the ICI and the control group (n = 60) at the time of their death or at any stage of the study.
//M
-
. ..Yzm--W~.__~
. . . . . . .. . . .. ..I I
O!
72
48
f 96
84 tlours of Exposure
-
Control
---
ICI (0 I yM/kg/hr)
‘..
.. ICI (IOyM/kg/hr)
Fig. 4 A: 6-Keto-PGF,,, the stable metabolite of PGI,, in pg/ml vs hours of exposure to >95% oxygen. B: TXB,, the stable metabolite of TXA,, in pg/ml vs hours of exposure to >95% oxygen. The bars represent standard errors of the mean.
Figures 4A and B summarize BAL fluid levels of cyclooxygenase products 6-keto-PGFi, vs hours of hyperoxic exposure and TXB2 vs hours of hyperoxic exposure. They were, as were the total protein and polymorphonuclear cell influx, relatively low after 48 and 72 h of oxygen exposure. After 84 and 96 h of hyperoxia both of these products increased in BAL fluid. Again, there were no significant differences between the three groups at any stage of the study. Figure 5 shows the mortality rates in relation to the duration of exposure to hyperoxia of ICI and control animals. 50% mortality was found to be at 72 h of hyperoxic exposure. There were no significant differences at any time between the ICI and the control group. The rabbits were also weighed daily to determine if their mother was showing preference for either the ICI or control animals and to see if the drug itself was affecting their development. There was no
MORTALITY % mortality 1001 80’
84
60 Hours -
Control
---
ICI (0
I
108
7
132
of Exposure
I rrU/kg/hr)
Fii. 5 The percent mortality of the rabbits at any given number of hours of exposure to >95% oxygen (ICI) vs hours of exposure to >95% oxygen (control).
Our data demonstrate that the administration of a specific leukotriene D4 and E4 receptor antagonist to oxygen susceptible, maturing, 7-day-old rabbits does not have a protective effect against the development of oxygen induced lung injury. Specifically, the administration of this compound did not inhibit PMN influx, the development of proteinaceous pulmonary edema, or improve survival with prolonged oxygen exposure. Several possible mechanisms may have contributed to these results: 1. Oxygen radicals by themselves are very toxic to the lung and their unopposed actions could play a significant and primary role in hyperoxic lung injury. 2. Other biochemical and cellular mediators, e.g. LTB4, neutrophils, macrophages, and lipoxins, may play a greater role than previously appreciated in in vitro experiments. 3. The drug did not inhibit the actions of LTD,+. The free radical theory of oxygen toxicity has generally been accepted as the biochemical and molecular mechanism that best explains injury secondary to hyperoxia (26). Oxygen radicals alone may cause sufficient injury to induce mortality by inactivating enzymes, peroxidizing membrane double bonds, causing alterations in membranes, cross-linking and causing schisms of DNA fragments, and causing sulfhydryl oxidation of protein moieties (26). The superoxide radical itself has been shown to damage and kill cells (27-30). Oxygen induced injury to endothelial and epithelial barriers of alveolar walls can cause pulmonary edema (26). All these effects, by themselves or in combination, may be sufficient to cause extensive injury and even mortality. The preceding paragraph may in part explain why the administration of a LTD4 and LTE4 antagonist did not inhibit the development of pulmonary edema. A difficulty in examining oxygen induced lung injury is that phenomena that occur in vitro may not be important or may not occur in vivo. Lipoxygenase products have been shown to increase systemic vascular permeability (31-33) and appear to increase pulmonary vascular permeability in a number of species including the rabbit (9, 11, 19, 34-36). This was further supported when Farrukh et al (19) showed in an in vitro experiment that the administration of LTD4 caused an increase in pulmonary vascular permeability in rabbits. While LTD4 may be one of the main mediators of pul-
LT Receptor Antagonist and Prevention of Hyperoxic Lung Injury
monary edema and oxidant induced pulmonary injury in vitro, there may be many other cellular and biochemical mediators at work in the intact animal that may not be evident in vitro. While the administration of ICI may have completely blocked the effects of LTD4, other unopposed mediators may be sufficient to produce pulmonary edema and lung injury. An important cell and source of mediators that may not be evident in in vitro experiments is the PMN. Polymorphonuclear leukocytes may play an important role in regulating fluid efflux from venules. This may imply that potent chemoattractant leukotrienes, e.g. LTB4, may also indirectly increase tissue edema through the recruitment of neutrophils (37). While ICI 198,615 is a leukotriene receptor antagonist with a high affinity for LTD4 and LTE4 (38), it has no effect against LTB4. LTB4 is one of the most potent chemokinetic and chemotactic agents known, able to affect the human, rat and rabbit PMN. It has therefore been proposed that LTB4 has a role in leukocyte recruitment during inflammation (31, 39, 40). The accumulation of neutrophils has been further postulated to contribute to lung injury during hyperoxia (37). It was also found that the exposure of rats to hyperoxia caused a marked increase in the chemoattractant activity of BAL fluid that was correlated with the increase in the number of PMNs in lung ravages. These events were followed by the death of the animal in a few hours (41). White cells, including macrophages, also actively participate in the further generation of active oxygen species (42), e.g. the superoxide anion, hydrogen peroxide and the hydroxyl radical (43-45), which can damage and kill cells. Leukotoxins, metabolites of the 1Slipoxygenase pathway, may also contribute to oxidant induced lung injury. Phospholipase A2 has been shown to cause the release of these products in addition to the leukotrienes via the release of the precursors of arachidonic acid. These products have been isolated from lung lavages of rats breathing pure oxygen and have been shown to have an uncoupling effect of mitochondrial respiration (46). Cyclooxygenase products in the current study were not increased by the ICI-mediated inhibition of LT function. The fact that 50% mortality occurred at 72 h in our studies was somewhat surprising. At that point i.n time only the lung wet weight to body weight ratios had begun to increase. All other indicators of injury in the lung, e.g. total protein, cyclooxygenase products, and neutrophil influx (Figs 1, 2 & S), had not shown any change from the 48-h measurement. This can be interpreted to mean that pulmonary edema in the absence of any other injury may be sufficient to cause mortality. It may also mean that the severe direct toxic
163
effects of oxygen radicals and neutrophils to the cells themselves were also enough to result in mortality. Oxygen may also be toxic to other organ systems so that the time of mortality is not based solely on sufficient damage to the lungs. In conclusion, the administration of a leukotriene D4 and E$ receptor antagonist was insufficient to stop the formation of pulmonary edema, attenuate mortality, or attenuate pulmonary injury secondary to hyperoxia. This experiment suggests that a number of other mediators may be involved in lung injury and that the inhibition of only one arm of the arachidonic acid cascade may not be sufficient to either prevent or attenuate oxygen induced injury. Acknowledgements Supported in part by the Dee and Moody Research Fund of Evanston Hospital. Presented in part at the Society for Pediatric Research, May 1989.
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