Modulation of Neurogenic Inflammation in Rat Trachea A .A . Norris,* M .E . Leeson, D .M . Jackson, M .C . Holroyde Department of Pharmacology, Fisons plc, Research and Development Laboratories, Bakewell Road, Loughborough, Leicestershire, LE11 ORH, UK SUMMARY . Antidromic stimulation of the cervical vagus nerve in anaesthetised rats resulted in plasma protein extravasation in the trachea . This response was potentiated significantly by pretreatment of the animals with an enkephalinase inhibitor, thiorphan (100 pg/kg) . Exposure of animals to ozone (5-6 ppm) for 30 min caused a significant increase in the numbers of lung lavage epithelial cells but failed to potentiate neuronally-evoked tracheal oedema . Several compounds were investigated for anti-permeability effects on thiorphan-pretreated, nerve stimulated animals. Morphine (3 mg/kg) and salbutamol (100 pg/kg) were the most efficacious agents used and resulted in 66 ± 14% and 61 ± 9% inhibition of tracheal oedema, respectively . Sodium cromoglycate at 30 mg/kg produced a small but significant reduction in oedema (34±10%) . Dexamethasone (3 mg/kg), methysergide (2 mg/kg) and theophylline (10 mg/kg) did not affect neurogenic oedema .
INTRODUCTION
METHODS
It is well established that a neurogenic inflammation can be elicited in the central airway of rats and guinea-pigs by antidromic stimulation of the sensory nerves in the cervical vagus .'-' The inflammatory response is associated with increased vascular permeability, leukocyte infiltration of the airways and increased mucus production .' Stimulation of sensory Cfibres is known to release a number of peptides of the tachykinin family such as substance P and neurokinin A . 4 It has been shown specifically in the isolated lungs of guinea-pigs that vagal nerve stimulation releases substance P and neurokinin A into the perfusing solutions and that these neuropeptides are capable of inducing airway oedema in guinea-pigs .' Although not proven, there may be a significant neurogenic inflammatory component in airway inflammation associated with bronchial asthma .' It is possible therefore that currently used anti-asthmatic agents might exert their therapeutic effects in part by suppressing neurogenic inflammation in the airways in man . The purpose of the present study was to investigate whether neuronally-evoked oedema in rat airways could be affected by a range of therapeutic agents . Since preliminary studies demonstrated only a small increase in vascular leakage after nerve stimulation, it was also necessary to explore mechanisms for potentiating the neurogenic response prior to drug evaluation .
Male CR/CD rats (200-300 g) in groups of 5 or 6 were anaesthetised with sodium pentobarbitone (60 mg/ kg/ip) and were allowed to respire spontaneously . The right jugular vein and right carotid artery were cannulated for administration of drugs and for measurement of blood pressure, respectively . Both cervical vagi were isolated and cut and the caudal ends were mounted on platinum electrodes immersed in liquid paraffin . Atropine (1 mg/kg) and Evans blue dye (2 .0%) were administered as single i .v . injections 5 min prior to stimulation of the vagi . Thiorphan (100 pg/kg) (an enkephalinase inhibitor) was administered 10 min prior to atropine in some experiments . Drugs were administered i .v . between 1 and 5 min before stimulation or s .c . 18 h before, in the case of dexamethasone . Nerve stimulation was carried out for 5 min at 40 Hz/5 msec/10 V . Sham controls received the same treatment as above except for nerve stimulation . Animals were killed 10 min after the stimulation period was completed . The whole trachea was dissected out and placed in 1 ml of formamide for 24 h to extract the dye . The content of dye in the tissues was estimated by spectrophotometry . The optical density of the samples was read at 620 nm and was compared with those of standard concentrations of Evans blue dye . The concentration of dye in the tracheas was expressed as ng per mm tracheal length . In a separate series of experiments, to investigate a means of potentiating the neurogenic response, groups of 5 conscious rats were exposed to ozone (5-6 ppm) for 30 min in a perspex chamber . Ozone was manufactured by passing dry air through an ozone generator (Type BA 023012 ; Wallace & Tier-
*Correspondence to : A .A. Norris, Department of Pharmacology, Fisons plc, Research and Development Laboratories, Bakewell Road, Loughborough, Leicestershire LEI I ORH, UK . 180
Modulation of Neurogenic Inflammation in Rat Trachea
nan, Tonbridge, Kent .) as described previously .' The concentration of ozone within the exposure chamber was monitored continuously with an ultraviolet ozone detector (Dasibi Model 1008-AH ; Glendale, California, USA .) . Ozone treated animals were used in vagal stimulation studies, as described above, 90 min and 150 min after the exposure was completed . Bronchoalveolar lavage (BAL) was carried out on different groups of rats exposed to ozone at the times indicated above, in order to quantitate and confirm that cellular changes occurred within the lungs after ozone . Animals were killed with sodium pentobarbitone (200 mg/kg/ip) and the tracheas cannulated . The lungs were lavaged with 2 x 5 ml aliquots of phosphate buffered saline and the fluid recovered by suction (approximately 80% recovery) . Each lavage sample was centrifuged at 1000 rpm for 10 min at 4°C . The cell pellet was resuspended in 0 .5 ml Hank's buffer and a 50 .t aliquot diluted with 450 tl Kimura's stain .' Cells were counted in a modified FuchsRosenthal haemocytometer . The following drugs were used : atropine sulphate, thiorphan, salbutamol sulphate, theophylline (each from Sigma), sodium pentobarbitone (May & Baker), dexamethasone sodium phosphate (Organon Ltd), sodium cromoglycate (Fisons plc), morphine sulphate (Richard Daniel & Son Ltd, Derby) . Evans blue dye was purchased from BDH . All drugs were dissolved in saline except thiorphan which was dissolved in ethanol followed by dilution in saline . Data analysis : All data were expressed as arithmetic means ± SEM . Statistical analyses were performed using a Mann-Whitney U test .
RESULTS Initial experimental results showed only a modest but significant (p < 0 .05) increase in dye extravasation in the tracheas after nerve stimulation (2 .07±0.27 times sham control; n = 4 experiments) (Fig . 1) . Further experiments revealed that pretreatment of animals with thiorphan (100 .tg/kg) significantly (p < 0 .05) potentiated the neurogenic response (3 .26±0 .18 times sham control ; n = 5 experiments) (Fig . 1) whereas exposure to ozone had no effect on protein extravasation caused by vagal stimulation (2 .08±0 .2 times sham control after 90 min ; 2 .61 ±0 .26 times sham control after 150 min) (Fig . 2) . Preliminary experiments revealed that ozone alone did not cause a tracheal oedema . However, ozone pretreatment caused a significant (p < 0 .05) increase in the numbers of epithelial cells recovered by BAL (Fig. 3), which indicates that structural damage had occurred within the airways . The evaluation of compounds was carried out in animals that received thiorphan pretreatment . The results were shown in Figure 4 . Morphine (3 mg/kg)
600,
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Fig . 1-Effects of vagal nerve stimulation on Evans blue dye extravasation in rat trachaea in the absence (control) and presence of thiorphan (100 ug/kg) . Open columns, sham controls; hatched columns, vagal nerve stimulation . Each column represents mean ± SEM (n = 6) . Statistical differences (p < 0 .05) are shown between sham and vagal nerve stimulated animals, for both thiorphan and non-thiorphan treated groups( •) and
between vagal nerve stimulated animals only, in the absence and presence of thiorphan (t) . and salbutamol (100 µg/kg) were the most efficacious agents used, reaching 66± 14% and 61 ±9% inhibition of oedema respectively . Higher doses of morphine (10 mg/kg) caused respiratory depression and the animals died before completion of the time course . The anti-asthmatic agent sodium cromoglycate given at 30 mg/kg attenuated the oedematous response by a small (34± 10%) but significant amount (Fig . 4) . In contrast dexamethasone (3 mg/kg), theophylline (10 mg/kg) and methysergide (2 mg/kg) had no effect on neurogenic oedema (Fig . 4) .
DISCUSSION Initial experiments were designed to optimise the neurogenic oedema prior to evaluation of the drugs . Substance P which is likely to be one of the mediators involved in the neurogenic response' is known to be destroyed by the neutral metalloendopeptidases (NEP) or enkephalinase 10 located within airway epithelial cells ." Experiments were carried out therefore to inhibit the enzyme using thiorphan, an agent shown previously to potentiate substance P-mediated bronchoconstriction in vitro 12 and in vivo' 3 and also to investigate the effects of ozone exposure on neurogenic inflammation . Although ozone is known to cause peripheral lung damage 14 characterised by deepithelialisation, minor effects have also been detected in the trachea, notably goblet cell discharge .) s However, in one study 16 bronchial hyperreactivity to substance P was reported in ozone treated guineapigs, without any gross damage to the airway epithel-
182
Pulmonary Pharmacology
400 Co 4) Z 0
2 300 E E
200_
s o 0_ Treatment
Air
Ozone
Ozone
Time after exposure
90min
90min
150min
Fig. 2-Effects of exposure of animals to air or ozone (5-6 ppm) for 30 min on vagal stimulation-induced extravasation of Evans blue dye into rat tracheas. Animals were used 90 min or 150 min after exposure to ozone and 90 min after exposure to air . Open columns, sham controls; hatched columns, nerve stimulation . Each column represents mean ± SEM (n = 6) . A statistically significant difference (p < 0 .05) between sham controls and nerve stimulated animals is shown by an asterisk .
10 E IT
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Air
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Ozone
Time after exposure
90min
90min
150min
Fig. 3-Effects of exposure of animals to air or ozone (5-6 ppm) for 30 min on lung lavage epithelial cells . Lungs were lavaged 90 min or 150 min after ozone and 90 min after air . Each column represents means ± SEM (n = 5) and statistical significance (p < 0 .05) between air and ozone treatments is indicated by an asterisk .
ium and this was taken to indicate a functional impairment of enkephalinase . There is no evidence from the present study to indicate a similar effect on tracheal enkephalinase, since exposure to ozone did not potentiate the neuropeptide-mediated inflammatory response,4 but was capable of removing significant numbers of epithelial cells, presumably from the distal airways . An increase in neurogenic inflammation of about
50% occurred after pretreatment with thiorphan and this is in keeping with the in vivo activity of this agent described by Shore et al ." Subsequently, thiorphan pretreatment was used in all studies in which drugs were evaluated . The potent inhibition of tracheal oedema seen with morphine agrees with previous studies ."' It is likely that this effect occurs through activation of presynaptic opiate receptors resulting in inhibition of neuropeptide release from sensory nerves ." This mechanism has also been proposed for opioid effects on noncholinergic vagally-mediated bronchoconstriction in guinea-pigs .' 9 In contrast, the anti-leakage effects of salbutamol are thought to be mediated by a relaxant effect on venular endothelial cells via B 2 adrenoceptors . 20 This mechanism confers an anti-permeability effect on B2 agonists against oedema induced by nerve stimulation or by directly acting agonists, e .g. histamine, and distinguishes this category of compounds from the opiates . It is not certain whether this effect is evident in asthma , 21 however, it is noteworthy that salbutamol has been reported to reduce lung endothelial permeability to plasma proteins in adult respiratory distress syndrome .22 Sodium cromoglycate has been shown previously to inhibit mediator-induced leakage of plasma proteins into and across the airway walls of guinea-pigs 23 and this activity has been forwarded as a contributory factor in its clinical efficacy.21 This finding is supported by the present studies on neurogenic inflammation in rats. The mechanism for this activity is not clear, but it is unlikely to involve inhibition of release of mast cell mediators, since methysergide was inactive in the model . It is known that sodium cromoglycate, at least, is capable of suppressing bronchial c-
Modulation of Neurogenic Inflammation in Rat Trachea
1 83
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Fig. 4-Effects of compounds on neurogenic oedema induced in the presence of thiorphan (100 ug/kg) in rat tracheas . Each column represents mean ± SEM (n = 6) . A significant difference (p < 0 .05) between vehicle controls and drug-treated animals is indicated by an asterisk .
fibre reflex activity in animals ."' Furthermore, it has been proposed that the anti-asthmatic activity of these compounds is related to inhibition of axon reflex mechanisms ." This may well represent a 'morphinelike' action resulting in inhibition of neuropeptide release from afferent neurones . However, it has also been shown recently that sodium cromoglycate acts as a tachykinin receptor antagonist in human skin26 and this activity may be important in the present studies . Theophylline produced only a small but not significant inhibition of neurogenic oedema, in contrast to the marked activity seen with this compound (and with another xanthine, enprofylline) in a model of guinea-pig tracheal permeability ." The precise reason for this variance is unknown although other studies have also been unable to show anti-permeability activity with xanthines ." The inactivity of a steroid, dexamethasone in the present studies is surprising and contrasts with its potent anti-permeability effects in skin lesions, induced by mediators and antigen challenge . 28 However, it has been noted that a difference in steroid sensitivity exists at various anatomical sites .2' For example, dexamethasone is capable of preventing platelet-activating factor induced oedema in the paw but not in the pleural cavity of rats .29 We have also been unable to demonstrate an anti-permeability effect of dexamethasone against mediator-induced tracheal permeability in rats (unpublished findings) . The clinical significance of the findings with dexamethasone is unclear . It may indicate that neurogenic inflammation does not contribute to the airway oedema in asthma, alternatively, it may occur during a phase in which steroids are ineffective e.g . immediately post antigen challenge ."
In conclusion, a consistent neurogenic inflammation was induced in rat tracheas only if enkephalinase was inhibited. Although it is not known whether asthmatic airways possess a similar `biochemical abnormality', it is possible that such a feature exists and may distinguish asthmatics from normals . References 1 . Lundberg J M, Saria A . Capsaicin-sensitive vagal neurons involved in control of vascular permeability in rat trachea. Acta Physiol Scand 1982 ; 115 : 521-523 . 2 . Lundberg J M, Saria A, Brodin E, Rosell S, Folkers K A . Substance P antagonist inhibits vagally induced increase in vascular permeability and bronchial smooth muscle contraction in the guinea-pig . Proc Natl Acad Sci 1983 ; 80 : 1120-1124. 3 . McDonald D M . Neurogenic inflammation in the respiratory tract : Actions of sensory nerve mediators on blood vessels and epithelium of the airway mucosa . Am Rev Resp Dis 1987 ; 136 : S65-S72 . 4. Hua X Y, Theodorsson-Norheim E, Brodin E, Lundberg J M . Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in the capsaicin sensitive sensory neurons in the guinea-pig . Regulatory Peptides 1985 ; 13 : 1-19 . 5 . Saria M, Martling C-R, Yan Z, TheodorssonNorheim E, Gamse R, Lundberg J M . Release of multiple tachykinins from capsaicin sensitive sensory neurones in the lung by bradykinin, histamine, dimethylphenyl piperazinium and vagal nerve stimulation . Am Rev Resp Dis 1988 ; 137 : 1330-1335 . 6 . Rogers D F, Belvisi M G, Aursudkij B, Evans T W, Barnes P J . Effects and interactions of sensory neuropeptides on airway microvascular leakage in guinea-pigs . Br J Pharmac 1988; 95: 1109-1116 . 7. Barnes P J. Airway neuropeptides and asthma . TIPS 1987; 8 : 24-27 . 8 . Holroyde M C, Norris A A . The effects of ozone on reactivity of upper and lower airways in guinea-pigs . Br J Pharmac 1988; 94: 938-946 . 9 . Kimura I, Moritani Y, Tanizaki Y . Basophils in bronchial asthma with reference to reagin-type allergy . Clin Allergy 1973 ; 3 : 195-202.
184 Pulmonary Pharmacology 10 . Stimler-Gerard N P . Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea-pig lung . J Clin Invest 1987; 79 : 1819-1825 . 11 . Advenier C, Devillier P, Matran R, Naline E . Influence of epithelium on the responsiveness of guinea-pig isolated trachea to adenosine . Br J Pharmac 1988 ; 93 : 295-302 . 12. Devillier P, Advenier C, Drapeau G, Marsac J, Regoli D . Comparison of the effects of epithelium removal and of an enkephalinase inhibitor on the neurokinininduced contractions of guinea-pig isolated trachea . Br J Pharmac 1988 ; 94 : 675-685 . 13 . Shore S A, Stimler-Gerard N P, Coats S R, Drazen J M . Substance P-induced bronchoconstriction in the guinea-pig . Am Rev Resp Dis 1988 ; 137 : 331-336. 14. Plopper C G, Dungworth D L, Tyler W S . Pulmonary lesions in rats exposed to ozone . Am J Pathol 1973 ; 71 : 375-394 . 15 . Murlas C G, Rouson J H . Sequence of pathological changes in the airway mucosa of guinea-pigs during ozone induced bronchial hyperreactivity. Am Rev Resp Dis 1985 ; 131 : 314-320 . 16 . Yeadon M, Wilkinson D, Payne A N . Ozone induces bronchial hyperreactivity to inhaled substance P by functional inhibition of enkephalinase . Proc Br Pharm Soc 1990; P35 . 17 . Belvisi M G, Rogers D F, Barnes P J . Neurogenic plasma extravasation: Inhibition by morphine in guinea-pig airways in vivo . J Appl Physiol 1989; 66 : 268-272. 18 . Bartho I, Szolcsani J . Opiate agonists inhibit neurogenic plasma extravasation in the rat . Eur J Pharmac 1981 ; 73 : 101-104 . 19 . Belvisi M G, Chung K F, Jackson D M, Barnes P J . Opioid modulation of non-cholinergic neural bronchoconstriction in guinea-pig in vivo . Br J Pharmac 1988; 95 : 413-418 . 20 . Persson C G A, Svensjo E . Vascular responses and their suppression : drugs interfering with venular permeability . In : Bonta I L, Bray M A, Parnham M J, eds . Handbook of inflammation . Volume 5 : The pharmacology of inflammation . Amsterdam : Elsevier, 1985 : 61-82 .
21 . Persson C G A . Cromoglycate, plasma exudation and asthma . TIPS 1987 ; 8 : 202-203 . 22 . Basran G S, Hardy J G, Woo S P, Ramasubramanian R, Byrne A J . Beta-2-adrenoceptor agonists as inhibitors of lung vascular permeability to radiolabelled transferrin in the adult respiratory distress syndrome in man . Eur J Nucl Med . 1986 ; 12 : 381-384 . 23 . Erjefalt I, Persson C G A . Anti-asthma drugs attenuate inflammatory leakage of plasma into airway lumen . Acta Physiol Scand . 1986; 128 : 653-654 . 24 . Dixon M, Jackson D M, Richards I M . The action of sodium cromoglycate on c-fibre endings in the dog lung. Br J Pharmac. 1980; 70 : 11-13 . 25 . Barnes P J. Asthma as an axon reflex . Lancet 1986 ; i : 242-245 . 26 . Crossman D C, Fuller R W . Inhibition of tachykinininduced wheal by sodium cromoglycate in human skin . Br J Clin Pharmac 1989 ; 27 : 710-711 P . 27 . Svensjo E, Roempke K . Microvascular aspects on edema formation and its inhibition by B 2 -receptorstimulants and some other anti-inflammatory drugs . In : Courtice F C, Garlick D G, Perry M A eds . Progress in microcirculation research II . Committee in Postgraduate Medical Education, University of New South Wales, Sydney, 1984 : 449-459. 28 . Inagaki N, Miura T, Nagai H, Koda A . Inhibitory effects of glucocorticoids on increased vascular permeability caused by passive cutaneous anaphylaxis and some chemical mediators in rats . Jap J Pharmac . 1988 ; 46 : 189-192 . 29 . Peers S H . Dexamethasone inhibits platelet activating factor-induced inflammation in the paw but not in the pleural cavity of rats . Eur J Pharmac . 1988 ; 150 : 131-135 . 30 . Pepys J, Davies R J, Breslin A B X . The effect of inhaled beclomethasone dipropionate (Becotide) and sodium cromoglycate on asthmatic reactions to provocation tests . Clin Allergy . 1974 ; 4 : 13-24. Date received : 27 July 1989 Date revised : 3 October 1989 Date accepted : 3 March 1990
INTRODUCTION
METHODS
It is well established that a neurogenic inflammation can be elicited in the central airway of rats and guinea-pigs by antidromic stimulation of the sensory nerves in the cervical vagus .'-' The inflammatory response is associated with increased vascular permeability, leukocyte infiltration of the airways and increased mucus production .' Stimulation of sensory Cfibres is known to release a number of peptides of the tachykinin family such as substance P and neurokinin A . 4 It has been shown specifically in the isolated lungs of guinea-pigs that vagal nerve stimulation releases substance P and neurokinin A into the perfusing solutions and that these neuropeptides are capable of inducing airway oedema in guinea-pigs .' Although not proven, there may be a significant neurogenic inflammatory component in airway inflammation associated with bronchial asthma .' It is possible therefore that currently used anti-asthmatic agents might exert their therapeutic effects in part by suppressing neurogenic inflammation in the airways in man . The purpose of the present study was to investigate whether neuronally-evoked oedema in rat airways could be affected by a range of therapeutic agents . Since preliminary studies demonstrated only a small increase in vascular leakage after nerve stimulation, it was also necessary to explore mechanisms for potentiating the neurogenic response prior to drug evaluation .
Male CR/CD rats (200-300 g) in groups of 5 or 6 were anaesthetised with sodium pentobarbitone (60 mg/ kg/ip) and were allowed to respire spontaneously . The right jugular vein and right carotid artery were cannulated for administration of drugs and for measurement of blood pressure, respectively . Both cervical vagi were isolated and cut and the caudal ends were mounted on platinum electrodes immersed in liquid paraffin . Atropine (1 mg/kg) and Evans blue dye (2 .0%) were administered as single i .v . injections 5 min prior to stimulation of the vagi . Thiorphan (100 pg/kg) (an enkephalinase inhibitor) was administered 10 min prior to atropine in some experiments . Drugs were administered i .v . between 1 and 5 min before stimulation or s .c . 18 h before, in the case of dexamethasone . Nerve stimulation was carried out for 5 min at 40 Hz/5 msec/10 V . Sham controls received the same treatment as above except for nerve stimulation . Animals were killed 10 min after the stimulation period was completed . The whole trachea was dissected out and placed in 1 ml of formamide for 24 h to extract the dye . The content of dye in the tissues was estimated by spectrophotometry . The optical density of the samples was read at 620 nm and was compared with those of standard concentrations of Evans blue dye . The concentration of dye in the tracheas was expressed as ng per mm tracheal length . In a separate series of experiments, to investigate a means of potentiating the neurogenic response, groups of 5 conscious rats were exposed to ozone (5-6 ppm) for 30 min in a perspex chamber . Ozone was manufactured by passing dry air through an ozone generator (Type BA 023012 ; Wallace & Tier-
*Correspondence to : A .A. Norris, Department of Pharmacology, Fisons plc, Research and Development Laboratories, Bakewell Road, Loughborough, Leicestershire LEI I ORH, UK . 180
Modulation of Neurogenic Inflammation in Rat Trachea
nan, Tonbridge, Kent .) as described previously .' The concentration of ozone within the exposure chamber was monitored continuously with an ultraviolet ozone detector (Dasibi Model 1008-AH ; Glendale, California, USA .) . Ozone treated animals were used in vagal stimulation studies, as described above, 90 min and 150 min after the exposure was completed . Bronchoalveolar lavage (BAL) was carried out on different groups of rats exposed to ozone at the times indicated above, in order to quantitate and confirm that cellular changes occurred within the lungs after ozone . Animals were killed with sodium pentobarbitone (200 mg/kg/ip) and the tracheas cannulated . The lungs were lavaged with 2 x 5 ml aliquots of phosphate buffered saline and the fluid recovered by suction (approximately 80% recovery) . Each lavage sample was centrifuged at 1000 rpm for 10 min at 4°C . The cell pellet was resuspended in 0 .5 ml Hank's buffer and a 50 .t aliquot diluted with 450 tl Kimura's stain .' Cells were counted in a modified FuchsRosenthal haemocytometer . The following drugs were used : atropine sulphate, thiorphan, salbutamol sulphate, theophylline (each from Sigma), sodium pentobarbitone (May & Baker), dexamethasone sodium phosphate (Organon Ltd), sodium cromoglycate (Fisons plc), morphine sulphate (Richard Daniel & Son Ltd, Derby) . Evans blue dye was purchased from BDH . All drugs were dissolved in saline except thiorphan which was dissolved in ethanol followed by dilution in saline . Data analysis : All data were expressed as arithmetic means ± SEM . Statistical analyses were performed using a Mann-Whitney U test .
RESULTS Initial experimental results showed only a modest but significant (p < 0 .05) increase in dye extravasation in the tracheas after nerve stimulation (2 .07±0.27 times sham control; n = 4 experiments) (Fig . 1) . Further experiments revealed that pretreatment of animals with thiorphan (100 .tg/kg) significantly (p < 0 .05) potentiated the neurogenic response (3 .26±0 .18 times sham control ; n = 5 experiments) (Fig . 1) whereas exposure to ozone had no effect on protein extravasation caused by vagal stimulation (2 .08±0 .2 times sham control after 90 min ; 2 .61 ±0 .26 times sham control after 150 min) (Fig . 2) . Preliminary experiments revealed that ozone alone did not cause a tracheal oedema . However, ozone pretreatment caused a significant (p < 0 .05) increase in the numbers of epithelial cells recovered by BAL (Fig. 3), which indicates that structural damage had occurred within the airways . The evaluation of compounds was carried out in animals that received thiorphan pretreatment . The results were shown in Figure 4 . Morphine (3 mg/kg)
600,
1 81
't
m
r
500
E
400
2 300 . a C LU
200 . 100. 0Control
Thiorphan
Fig . 1-Effects of vagal nerve stimulation on Evans blue dye extravasation in rat trachaea in the absence (control) and presence of thiorphan (100 ug/kg) . Open columns, sham controls; hatched columns, vagal nerve stimulation . Each column represents mean ± SEM (n = 6) . Statistical differences (p < 0 .05) are shown between sham and vagal nerve stimulated animals, for both thiorphan and non-thiorphan treated groups( •) and
between vagal nerve stimulated animals only, in the absence and presence of thiorphan (t) . and salbutamol (100 µg/kg) were the most efficacious agents used, reaching 66± 14% and 61 ±9% inhibition of oedema respectively . Higher doses of morphine (10 mg/kg) caused respiratory depression and the animals died before completion of the time course . The anti-asthmatic agent sodium cromoglycate given at 30 mg/kg attenuated the oedematous response by a small (34± 10%) but significant amount (Fig . 4) . In contrast dexamethasone (3 mg/kg), theophylline (10 mg/kg) and methysergide (2 mg/kg) had no effect on neurogenic oedema (Fig . 4) .
DISCUSSION Initial experiments were designed to optimise the neurogenic oedema prior to evaluation of the drugs . Substance P which is likely to be one of the mediators involved in the neurogenic response' is known to be destroyed by the neutral metalloendopeptidases (NEP) or enkephalinase 10 located within airway epithelial cells ." Experiments were carried out therefore to inhibit the enzyme using thiorphan, an agent shown previously to potentiate substance P-mediated bronchoconstriction in vitro 12 and in vivo' 3 and also to investigate the effects of ozone exposure on neurogenic inflammation . Although ozone is known to cause peripheral lung damage 14 characterised by deepithelialisation, minor effects have also been detected in the trachea, notably goblet cell discharge .) s However, in one study 16 bronchial hyperreactivity to substance P was reported in ozone treated guineapigs, without any gross damage to the airway epithel-
182
Pulmonary Pharmacology
400 Co 4) Z 0
2 300 E E
200_
s o 0_ Treatment
Air
Ozone
Ozone
Time after exposure
90min
90min
150min
Fig. 2-Effects of exposure of animals to air or ozone (5-6 ppm) for 30 min on vagal stimulation-induced extravasation of Evans blue dye into rat tracheas. Animals were used 90 min or 150 min after exposure to ozone and 90 min after exposure to air . Open columns, sham controls; hatched columns, nerve stimulation . Each column represents mean ± SEM (n = 6) . A statistically significant difference (p < 0 .05) between sham controls and nerve stimulated animals is shown by an asterisk .
10 E IT
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5-
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Treatment
Air
Ozone
Ozone
Time after exposure
90min
90min
150min
Fig. 3-Effects of exposure of animals to air or ozone (5-6 ppm) for 30 min on lung lavage epithelial cells . Lungs were lavaged 90 min or 150 min after ozone and 90 min after air . Each column represents means ± SEM (n = 5) and statistical significance (p < 0 .05) between air and ozone treatments is indicated by an asterisk .
ium and this was taken to indicate a functional impairment of enkephalinase . There is no evidence from the present study to indicate a similar effect on tracheal enkephalinase, since exposure to ozone did not potentiate the neuropeptide-mediated inflammatory response,4 but was capable of removing significant numbers of epithelial cells, presumably from the distal airways . An increase in neurogenic inflammation of about
50% occurred after pretreatment with thiorphan and this is in keeping with the in vivo activity of this agent described by Shore et al ." Subsequently, thiorphan pretreatment was used in all studies in which drugs were evaluated . The potent inhibition of tracheal oedema seen with morphine agrees with previous studies ."' It is likely that this effect occurs through activation of presynaptic opiate receptors resulting in inhibition of neuropeptide release from sensory nerves ." This mechanism has also been proposed for opioid effects on noncholinergic vagally-mediated bronchoconstriction in guinea-pigs .' 9 In contrast, the anti-leakage effects of salbutamol are thought to be mediated by a relaxant effect on venular endothelial cells via B 2 adrenoceptors . 20 This mechanism confers an anti-permeability effect on B2 agonists against oedema induced by nerve stimulation or by directly acting agonists, e .g. histamine, and distinguishes this category of compounds from the opiates . It is not certain whether this effect is evident in asthma , 21 however, it is noteworthy that salbutamol has been reported to reduce lung endothelial permeability to plasma proteins in adult respiratory distress syndrome .22 Sodium cromoglycate has been shown previously to inhibit mediator-induced leakage of plasma proteins into and across the airway walls of guinea-pigs 23 and this activity has been forwarded as a contributory factor in its clinical efficacy.21 This finding is supported by the present studies on neurogenic inflammation in rats. The mechanism for this activity is not clear, but it is unlikely to involve inhibition of release of mast cell mediators, since methysergide was inactive in the model . It is known that sodium cromoglycate, at least, is capable of suppressing bronchial c-
Modulation of Neurogenic Inflammation in Rat Trachea
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0
Fig. 4-Effects of compounds on neurogenic oedema induced in the presence of thiorphan (100 ug/kg) in rat tracheas . Each column represents mean ± SEM (n = 6) . A significant difference (p < 0 .05) between vehicle controls and drug-treated animals is indicated by an asterisk .
fibre reflex activity in animals ."' Furthermore, it has been proposed that the anti-asthmatic activity of these compounds is related to inhibition of axon reflex mechanisms ." This may well represent a 'morphinelike' action resulting in inhibition of neuropeptide release from afferent neurones . However, it has also been shown recently that sodium cromoglycate acts as a tachykinin receptor antagonist in human skin26 and this activity may be important in the present studies . Theophylline produced only a small but not significant inhibition of neurogenic oedema, in contrast to the marked activity seen with this compound (and with another xanthine, enprofylline) in a model of guinea-pig tracheal permeability ." The precise reason for this variance is unknown although other studies have also been unable to show anti-permeability activity with xanthines ." The inactivity of a steroid, dexamethasone in the present studies is surprising and contrasts with its potent anti-permeability effects in skin lesions, induced by mediators and antigen challenge . 28 However, it has been noted that a difference in steroid sensitivity exists at various anatomical sites .2' For example, dexamethasone is capable of preventing platelet-activating factor induced oedema in the paw but not in the pleural cavity of rats .29 We have also been unable to demonstrate an anti-permeability effect of dexamethasone against mediator-induced tracheal permeability in rats (unpublished findings) . The clinical significance of the findings with dexamethasone is unclear . It may indicate that neurogenic inflammation does not contribute to the airway oedema in asthma, alternatively, it may occur during a phase in which steroids are ineffective e.g . immediately post antigen challenge ."
In conclusion, a consistent neurogenic inflammation was induced in rat tracheas only if enkephalinase was inhibited. Although it is not known whether asthmatic airways possess a similar `biochemical abnormality', it is possible that such a feature exists and may distinguish asthmatics from normals . References 1 . Lundberg J M, Saria A . Capsaicin-sensitive vagal neurons involved in control of vascular permeability in rat trachea. Acta Physiol Scand 1982 ; 115 : 521-523 . 2 . Lundberg J M, Saria A, Brodin E, Rosell S, Folkers K A . Substance P antagonist inhibits vagally induced increase in vascular permeability and bronchial smooth muscle contraction in the guinea-pig . Proc Natl Acad Sci 1983 ; 80 : 1120-1124. 3 . McDonald D M . Neurogenic inflammation in the respiratory tract : Actions of sensory nerve mediators on blood vessels and epithelium of the airway mucosa . Am Rev Resp Dis 1987 ; 136 : S65-S72 . 4. Hua X Y, Theodorsson-Norheim E, Brodin E, Lundberg J M . Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in the capsaicin sensitive sensory neurons in the guinea-pig . Regulatory Peptides 1985 ; 13 : 1-19 . 5 . Saria M, Martling C-R, Yan Z, TheodorssonNorheim E, Gamse R, Lundberg J M . Release of multiple tachykinins from capsaicin sensitive sensory neurones in the lung by bradykinin, histamine, dimethylphenyl piperazinium and vagal nerve stimulation . Am Rev Resp Dis 1988 ; 137 : 1330-1335 . 6 . Rogers D F, Belvisi M G, Aursudkij B, Evans T W, Barnes P J . Effects and interactions of sensory neuropeptides on airway microvascular leakage in guinea-pigs . Br J Pharmac 1988; 95: 1109-1116 . 7. Barnes P J. Airway neuropeptides and asthma . TIPS 1987; 8 : 24-27 . 8 . Holroyde M C, Norris A A . The effects of ozone on reactivity of upper and lower airways in guinea-pigs . Br J Pharmac 1988; 94: 938-946 . 9 . Kimura I, Moritani Y, Tanizaki Y . Basophils in bronchial asthma with reference to reagin-type allergy . Clin Allergy 1973 ; 3 : 195-202.
184 Pulmonary Pharmacology 10 . Stimler-Gerard N P . Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea-pig lung . J Clin Invest 1987; 79 : 1819-1825 . 11 . Advenier C, Devillier P, Matran R, Naline E . Influence of epithelium on the responsiveness of guinea-pig isolated trachea to adenosine . Br J Pharmac 1988 ; 93 : 295-302 . 12. Devillier P, Advenier C, Drapeau G, Marsac J, Regoli D . Comparison of the effects of epithelium removal and of an enkephalinase inhibitor on the neurokinininduced contractions of guinea-pig isolated trachea . Br J Pharmac 1988 ; 94 : 675-685 . 13 . Shore S A, Stimler-Gerard N P, Coats S R, Drazen J M . Substance P-induced bronchoconstriction in the guinea-pig . Am Rev Resp Dis 1988 ; 137 : 331-336. 14. Plopper C G, Dungworth D L, Tyler W S . Pulmonary lesions in rats exposed to ozone . Am J Pathol 1973 ; 71 : 375-394 . 15 . Murlas C G, Rouson J H . Sequence of pathological changes in the airway mucosa of guinea-pigs during ozone induced bronchial hyperreactivity. Am Rev Resp Dis 1985 ; 131 : 314-320 . 16 . Yeadon M, Wilkinson D, Payne A N . Ozone induces bronchial hyperreactivity to inhaled substance P by functional inhibition of enkephalinase . Proc Br Pharm Soc 1990; P35 . 17 . Belvisi M G, Rogers D F, Barnes P J . Neurogenic plasma extravasation: Inhibition by morphine in guinea-pig airways in vivo . J Appl Physiol 1989; 66 : 268-272. 18 . Bartho I, Szolcsani J . Opiate agonists inhibit neurogenic plasma extravasation in the rat . Eur J Pharmac 1981 ; 73 : 101-104 . 19 . Belvisi M G, Chung K F, Jackson D M, Barnes P J . Opioid modulation of non-cholinergic neural bronchoconstriction in guinea-pig in vivo . Br J Pharmac 1988; 95 : 413-418 . 20 . Persson C G A, Svensjo E . Vascular responses and their suppression : drugs interfering with venular permeability . In : Bonta I L, Bray M A, Parnham M J, eds . Handbook of inflammation . Volume 5 : The pharmacology of inflammation . Amsterdam : Elsevier, 1985 : 61-82 .
21 . Persson C G A . Cromoglycate, plasma exudation and asthma . TIPS 1987 ; 8 : 202-203 . 22 . Basran G S, Hardy J G, Woo S P, Ramasubramanian R, Byrne A J . Beta-2-adrenoceptor agonists as inhibitors of lung vascular permeability to radiolabelled transferrin in the adult respiratory distress syndrome in man . Eur J Nucl Med . 1986 ; 12 : 381-384 . 23 . Erjefalt I, Persson C G A . Anti-asthma drugs attenuate inflammatory leakage of plasma into airway lumen . Acta Physiol Scand . 1986; 128 : 653-654 . 24 . Dixon M, Jackson D M, Richards I M . The action of sodium cromoglycate on c-fibre endings in the dog lung. Br J Pharmac. 1980; 70 : 11-13 . 25 . Barnes P J. Asthma as an axon reflex . Lancet 1986 ; i : 242-245 . 26 . Crossman D C, Fuller R W . Inhibition of tachykinininduced wheal by sodium cromoglycate in human skin . Br J Clin Pharmac 1989 ; 27 : 710-711 P . 27 . Svensjo E, Roempke K . Microvascular aspects on edema formation and its inhibition by B 2 -receptorstimulants and some other anti-inflammatory drugs . In : Courtice F C, Garlick D G, Perry M A eds . Progress in microcirculation research II . Committee in Postgraduate Medical Education, University of New South Wales, Sydney, 1984 : 449-459. 28 . Inagaki N, Miura T, Nagai H, Koda A . Inhibitory effects of glucocorticoids on increased vascular permeability caused by passive cutaneous anaphylaxis and some chemical mediators in rats . Jap J Pharmac . 1988 ; 46 : 189-192 . 29 . Peers S H . Dexamethasone inhibits platelet activating factor-induced inflammation in the paw but not in the pleural cavity of rats . Eur J Pharmac . 1988 ; 150 : 131-135 . 30 . Pepys J, Davies R J, Breslin A B X . The effect of inhaled beclomethasone dipropionate (Becotide) and sodium cromoglycate on asthmatic reactions to provocation tests . Clin Allergy . 1974 ; 4 : 13-24. Date received : 27 July 1989 Date revised : 3 October 1989 Date accepted : 3 March 1990
