Effect of cigarette smoke on bronch ial reactivity

Effect of cigarette smoke on bronchial reactivity. Respir 1995;2(4):231-235. OBJECTIVE: To compare the effects of a brid low level exposure to cigarette smoke in rats with known low (Sprague-Dawley) and high (Fisher) airway responsive ness. to test the hypothesis that airways reactivity influences the severity or duration of pulmonary function alterations after cigarette smoke exposure. METHODS: Baseline pulmonary function tests and metha choline dose response tests were conducted in IO Sprague Dawley and 10 Fisher rats. On the following day, the animals were reanaesthetized. and breathed for I min from a 2 L chamber into which 25 mL of fresh cigarette smoke had been injected, folluwed by a second set of pulmonary function and methacholine response tests: a final set was performed two weeks later. RESULTS: Sprague-Dawley rats were larger, with larger lung volumes, compliance and flow rates, but Fisher rats showed a fourfold higher airway reactivity. Although nci-ther resistance nor response to methacholine chanaed in either strain of animal, Sprague-Dawley rats showed an increase in residual volume post smoke, which was not sustained over two weeks. and sustained small increases in vital capacity, total lung capacity and static lung compli ance, with a sustained decrease in forced expiratory volume in 0.1 s. while Fisher rats showed only a small sustained increase in functional residual capacity. CONCLUSIONS: Although there are marked differences in pulmonary function between the two different strains of rats, increased airways responsiveness per se does not make the animal more sensitive to the acute effects of cigarctk~ smoke, and the effects of cigarette smoke on pulmonary function are not necessarily related to increased airway resistance. Pulmonary function allL~rations seen after brief cigarette smoke exposure may be sustained for a relatively long period of time. ( Pour

Rf. :. . <;n : fAT~ : Les nu , Sprague -Dawley c1aicn1 plus gros. avce des vo lumes pul monaircs. unc comp liancc (; l des debits plu~ g r,uid.,. B R()N(' I II A L II YPERIU,AC'T IVITY \IA Y BE A N IM!'Ql{ T ANT dctcrminant of d isease susccpt1bilily in ch ronic obstructive pulmonary di sease (C'OPD) ( 1.2). l .ong itudinal studic~ haVl' mdica!c d that airways hypcrrcactivily (3) and lac k of response to bronchodil,llors (J.4) arc import;1nl predicto rs ot ;111 incn:ased rate o f dee lme in fo rced expiratory volume in I s (FEV 1 ). indepe ndent of tltc c ffi:.'ch o f s mo king . S imilarl y. a recent study of young c iga rette smokers (5) has shown that Ilic prcsc 11cc of w hce;.ing. proh,ibl y ind1l·.1ti w of lung h ypcrresponsi vcness. is pn:dict ivc of a prog ress ive loss of vent i l;ito ry fun ction. A rece nt multice ntrc c linical tri a l ( Lun g He a lth St udy) ~ho,vcd th at current smokers with fun ct ional cvide nc e of early COPLJ had nons pecific airways hypcrrcspo ns ive ness 1ha1 was rdated to the baseline va lues fo r lun g funct ion (6). Tas hki n a nd co lleague s (7) have recent ly ~hown a dose -dependent effect of to bacco smok ing with airways responsiveness. whic h appears 10 be independent of the e ffec ts o f lung func ti o n. This latte r st udy wo uld suppon the concept that c:1gare!ll.'. smoke has a pri 1nilry e ffect o n airways h y pe rrc sponsivcncss.
Passive smoke exposure has also been re lated 10 increased ai rways responsi veness. both in c hildren at home (8) and in adults al the ir work place (9). Meclwnisms of induction uf airway re activ ity in these studies are unclear, but it has been suggested th;1t the proces~ may be rcla1.ed to an inllammalory response in the airways . A lth oug h, in ma n y animals . an ac ute expo sure to cigare tte smoke may be as soc ia ted w ith an im-rneJiatc increase in airways resistance ( l0-13) and ap pea rs to make the atrways more sensitive to st:1ndard doses of acC'lylc ho l i ne ( 14) or mcthac hol inc ( 13, I S). l he mcc han ism is disputcd.

ANIMALS AND METHODS
(irnu ps of 10 Sprag ue-Dawley ,llld 10 Fi sher rats we re used. On day I , baseline pulmo nary funct ion tests and m c thacho line c ha lle nge we re perfom1ed as docume nte d l~low: on day 2., the anima ls were re-a11acsthc1 i1,cd and were a llo w ed to breathe for I rn in from a 2. L c ham he r co11t;1i ning 25 ml fresh c igarctle smoke . This dose was c hosen frnm an initial st ud y us ing Sprague-Dawley rats that dc monstratcd that thi s smoke concentratio n wou ld produce a n inil ial rise in airways n::sistancc that would no t be sus taine d after 10 mins .
Ten minutes afte r the smoke inhalatio n. re peat pulmonary function tests and me 1hac h0line c hall enge w en: done. A fi nal set of pu lm onary fun c tio n te sts and mct hacholine challe nge w ere pe rfonned two weeks a ft er initia l ha sd inc va lues. To de term ine that rnethac ho line rcspo 11s ivcnc:--s c..lid not c ha nge d ue 10 the expcrimc nLa l procedure, a control gro up of fiv e Sprag ue-Da w ley rat s was studied us ing the s a m e protocol.
Pulmon  calculated as the diffe rence between mouth and pleural pressure. Resu lts were analyzed using the RA YTEC computergenerated pul monary function analysis system (Fine Science Too ls). Functional residual capacity (FRC) was measured by airway occlusion at end -ex piration, fo llowing which the rats were given supplementary doses of fentany l droperidol and were rendered apneic by hyperventilation. Pressure vol ume curves were constructed from values obtained by delhtion to -30 cm H20. in fl ation to +30 cm H20 and deflation to -30 cm H20. T wo such inflation-defl ation manoeuvres were perfonned before measureme nt of the curve (with concurrent calculation of lung volumes expiratory reserve vo lume, res idual volume. vital capacity and total lung capacity ITLC I).
Static lung compl iance was calculated between FRC and FRC plus 10 cm transpu lmonary pressure. A fl ow volume curve was constructed from va lues at inflation to +30 cm H20 and at rapid deflation at -50 cm H20 pressure. From this curve, forced vital capacity. forced expiratory fl ow. forced expiratory llow between 25 and 75% forced vital capac ity (FEF2. 1-75). FEYo. 1 and peak expiratory flo w were calculateJ . The !low vol ume curve was plotteJ by detem1i ning the flow at each of the volumes betwee n 30 and 95% TLC. The methac holine challenge was perfom1ed on spon taneous breathing rats. using a methodology adapted from Martin and colleagues (17.18). An Acorn nebu lizer (Trudell Med ical ) was used with 7 L/min air flow , which delivered the particles into a I L vented breathing chamber attached to the endotracheal tube. The rat was allowed to breathe spontaneously fro m this chamber for 2 mins, and after a 30 s period of room air breath ing, resistance was measured and averaged over approximately IO breaths. Base line resista nce was measured after de livery of norrn al saline; then doubling mcthac holine concentrations from 0.05 to 0.8 mg/mL were used. followed by concentrations increment ing by 0 .25 mg/rn L from I to 5 mg/mL. R200 was calcul ated from the dose response curve as the concentration of mcthacholine requ ired to dou ble base li ne resistance.
Following the pulmonary function tests. th an imal s were Can Respir J Vol 2 No 4 Winter 1995 kept warm, allowed to recover from the anaesthes ia anJ were ext ubated. They were housed in a lam inar hood in standard rat cages with paper pellets as bedding , and were allowed free access to rat chow and water. T he data were first nonnali,i;ed by log transformat ion. All analyses were done with the SYSTAT system (SYSTAT Inc. Illinois). Unpaired t tests were used to test for strain differences at each point in time. Paired t tests were performed to ascertain whether a change from baseline had occurred within an animal group. Two-way repeated measures analy s is of variance was used to compare level ol' pulmonary function both within and between the twn animal strains.
There was a marked di fference between the baseline values of the Sprague-Dawley and Fisher rats ( Table I) In the Sprague-Dawley rats, smoke produced an increase in resid ual volume (P<0.00 I). which did not pers ist ::ifter two weeks. This was associated with an increase in static lung compliance (P<0.03), and increases in vital capacity (P<0.04) and TLC (P<0.00 I ), with an upward shift in the pressure volume curve ( Pdl.00<1). all of which pers isted over two Although the expiratory flow volume curve was not altered after smoke, the FEVo.1 was significantly di minished (P<0.02), and there was a further decrease after two weeks (P<0.0 1 ).
In the Fishe r rats, the FRC was significant ly increased post smoke (P<0.001), and thi s change was persistent over the following two weeks (P<0.02). No other alterations in the lung volumes or pressure volume curve were found ; simila rly , the flow volume curve and flow rates remained stable.

DISCUSSION
We dev ised the present experiments to answer the fo llowing questions. First. are there any differences between the acute reactions of a nonnoreactive (Sprag ue-Daw ley) and a hyperreactive (Fisher) strain of rat to a m inimal dose of cigarette smoke? Second, is there any long term effect on the pulmonary fu nction or methacholine reactivity of exposure to a brief, low intensity, dose of cigarette smoke? We fou nd that, regardless of initial airways responsi veness, this mi nimal exposure did not increase sensitivity to methacholi ne. It did. however. alter pulmonary function, and severa l of these alterations were persistent. Surprising ly. the pulmonary function changes were primarily seen in the less responsive Sprague-Dawley animals. with only the FRC be ing altered in the Fisher rats.
In humans. short term inhalation of sidestream smoke will produce a dose-related increase in symptoms and a sign ificant decrease in pulmonary function in patients with increased methacholine sensitivity (1 9). Other stud ies in animal s have shown that cigarette smoke will increase airways resistance ( I 1-14 ). Xu and colleagues ( I 5) exposed rats to three cigarettes per day, five days per week, for 15 weeks. They fou nd that th is regimen did not al ter baseline res istance, but decreased the R200 and allowed the detection of a resista nce plateau in the methacholine dose response curve. Although they were able to demonstrate increases in TLC, they could not relate the changes in respo nsi veness to al terations of lung e lasticity. Our study suggests that changes in lung e lasticity, lung volumes and FEVo.1 may occur very early in the genes is of cigarette smoke-i nduced lung disea se, predating alterations in airway responsiveness. The nature of the alte rat ion of lung elasticity is unclear; although we have pre viously fou nd that exposure to cigarette smoke wi ll produce ini tial destruction o f lung co llagen matrix (20). the smoke exposure in that study was of much greater intensity and of longer duration than that of the present study . We do not consider it likely that the changes in pulmonary function shown in the present study can be attributed to the effects of inflam mation . lnduction of inflammation by the smoke would prefe rentially affect the airways and tx· reflected in increased resistance or increased bronc hial res pon siveness ( I 0, 14 ), changes not fou nd in our study. In support of this concl usion are the stud ies of Xu ct al ( 15) and Nishikawa and colleagues ( 13). Neither of these grou ps could demonstrate evidence for inflammation after a muc h more intensive exposure to c igarette smoke.
In humans, ai rway structure has bee n suggested to be an important predisposing fac tor for airways hyperrcsponsive.ness, and may e xplain the female sex bias fo r increased airways reac tivity in smokers with mil d COPD (2 1 ). In the present study, we foun d marked differences in the pulmonary funct ion of Sprague-Dawley and Fisher rats, all lung volumes and flo w rates bei ng hi gher in the former. Some of the differences may be att ri butable to differences in size as refl ected in the ir wei ght; Sprague-Dawley animal s weighed 475±39 g at baseline while the Fisher animals weighed 297± 17 g. The Fisher rats grew only 36±6 g compared with 72±6 g seen in the Sprague-Dawley animals. suggesting different unde rlyi ng growth rates. Possible differences in the lung structu re in the two strains could also explain the di fferences in pul mona ry function. Eidelman et al ( 16) showed that the Fisher rats have di ffe rent airway structure with increased amounts of airway smooth muscle compared with the less responsi ve Lew is rats . and suggested that they may explain the differe nces in innate airway responsiveness. It is also possible that these differences in lung structure could additionally explain the paucity of cigarette smoke-induced changes in pulmonary function in the Fisher compared with the Sprague-Dawley rats.

CONCLUSIONS
Although a brief, low intensity dose of cigarette smoke does not alter airways resistance or responsiveness, it does produce alterations in pulmonary function. some or which are