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AIRWA y RESPONSIVENFSS TO METHACHOLINE AND AIRWAY SMOOTH MUSCLE IN THE GUINEA PIG.
Dy
Anabelle M. Opazo Saez Department of Physiology
McGiII University, Montreal May, 1991
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the degree of
Masters of Science
® AlIabeUe M. Opazo Saez 1991
-~--------
ABSTRACT
The purpose of this stlldy was two-fold, 1) to examine the relation:ihip between the
amount of airway smooth muscle and the airway responsiveness to inhaled aerosolized
methacholine (MCh) If. gumea pigs, and 2) to characterize the distribution of airway
narrowing foIlowing MCh. Complete concentration-response curves were perform~ in
vivo in mechanically ventilated guinea pigs (n = 13). The response was determined by
measuring the lung re:.istance (RI.' cmH20/ml/s). The maximal response (Rmax), and
the concentration of MCh required to reach 50% of the maximal response, the ECso,
were th en compared with the amount of airway smooth muscle (ASM) measured by
morphometry. To examme the site and distribution of bronchoconstrictIOn, another group
of guinea pigs (n = 6) was prepared as above and exposed to aerosols of MCh until Rmax
was achieved. Then each animal was allowed to rerover to a pre-determined value along
its concentratIOn-response curve (100, 75, 60, 40, or 15 % of Rmax). A control animal
was aerosolized wlth saline. Alrway narrowing was given by the ratio of the airway
luminal area (LA) to the ideal lummal area (ILA).
The results showed considerable variability in the sensitivity (a 254 fold difference
in EC~o) but a small range (3.6 fold) of maximal responses. A substantial variability in
the quantity of ASM was found among airways of equivalent size. There was no
correlation between Rmax and ASM in the airways studied (n =429). However, there
was a negative correlation between the 10gECso and ASM (r=-.541, p<O.05) in
intraparenchymal cartilaginous alrways. The logEC5o also correlated weil with size of
intraparenchymaJ airways (r=O.72, p < 0.05). There was a heterogenolls distnbution of
bronchoconstnction In ail 'iized airways (n= 464), partIcularly 111 lh~ small airways
(diamder < 0.64 mm). The dominant site of constricti(\n to Meh was III the large
airways (diameter > 0.64 mm). There was a good correlatIon betwecn the coefficient
of variation of LA/ILA and the degree of constriction ln small (r=0.98) and large
airways (r=O.88). But there was no correlation bctwcen alrway narrow1l1g (LA/ILA)
and ASM. In the control animal the %SM shortenmg was about 20% across airways of
different si1',;s, regardless of the amount of ASM. ln contrast, the constnctcd lungs had
a %SM sh(, ,-1 ~ning that varied from 0 to 80% of the prcdlcted value for an unconstricted
airway, across dlfferent sized alrways wlth varying amount3 of ASM.
In summary, 1) the quantity of ASM docs not appear to detcrmine diffcrences in
maximal bronchoconstriction among normal guinea plgs; the Jack of a correlation
between responsiveness and amount of ASM may be explamed by the hcterogenous
distribution of bronchor.onstnction among the airways studled or the modahty of
challenge; 2) the sensltivity to Meh appears to be related to differences m the amount
of ASM in intraparenchymal cartilaginous !!.irways; 3) variabllity In the EC~[) may also
reflect differences in airway cross-sectional area; 4) Jung resistance appcars to be a good
measure of constriction since the morphometric measure of airway narrowmg correlatcd
weIl with resistance; 5) the heterogeneity of airway narrowing does not appear to he
determined by differences In ASM.
ii
RÉSUMÉ
Les objectifs vIsés par cette étude incluaient: 1) l'investigation chez le cobaye, de la
relation entre la quantité de muscles lisses (ASM) des voies respiratoires et la réponse
de celles-ci à l'mhalation de methacholine (MCh) en aérosol, ainsi que 2) la
détermination de la distribmion du rétrécissement des voies aériennes après provocation
à l'aide d'aérosols de MCh. Des courbes complètes de concentratiQn en fonction de la
réponse à la MCh ont été obtenues in vivo chez des cobayes (n = 13) ventilés à l'aide
d'un re~.plratcur. La réponse a été dcterminée par les mesures de résistance pulmonaire
(RI, cmH20/ml/s). La réponse maximale (Rmax) et la concentration de MCh requise
poUl' atteindre 50% de la réponse maximale (ECso) ont été comparées avec la quantité de
ASM mesurée par des méthodes morphométriques. Dans le but d'examiner les sites ainsi
que la dlstributK,.l de la bronchoconstnction, 6 cobayes ont été manipulés comme Cl-haut
mentionné et exposés à la MCh en aérosol jusqu'à ce que Rmax soit atteinte. Ensuite,
chaque ammal reposaIt jusqu'à ce qu'une valeur pré-établie de resistance sur sa courbe
concentratIOn-réponse soit attemte (100, 75, 60, 40 ou 15% de Rmax). Le control
recevait un aérosol de solution saline. Le ratio de la surface interne de la lumière (LA)
à la surface idéale de la lumière (ILA) a été utilisé comme indice dl~ rétrécissement des
voies aénennes.
Les résultats ont démontré une grande variabilité de la sensibilité (facteur de
variabilité de 254 pour ECso) mais une ressemblance chez les réponses :naximales
(facteur de variabiliié de 3.6 pour Rmax). Aussi, une variabilité importante a été
observée dans la quantité de ASM entre des voies respiratoires (0 =429) de grandeurs
similaires. Aucune corrélation n'a été P .aD lie entre Rmax et ASM pour les voies
repiratOires étudiées. Par contre, une corrélation négative a été trouvée entre le 10gECso
lU
et ASM (r=-O.541, p<0.05) dans les voies respiratoires cartilagineuses du parenchyme.
Une corrélation entre le logECso el les dimensions des voies aénennes parenchYl11ales a
aussi été établie (r=O.72, p<O.05». La distribution de la bronchoconstflctlon était
hétérogène dans les voies aériennes (n=464) de toute dimensIon, spécialement dans les
voies petites (rliamètre < 2mm). Le site le plus important de bronchoconstnctlOn duc à
la Meh était dans les voies respirat(\!~ès larges (diamètre> 2mm). Il y avait une bonne
corrélation entre le coefficier~ de variation LAIILA et le degré de constrictlon des vOies
respiratoires petites (r=0.90) et larges (r=0.88). Par contre, Il n'y avaIt pas de
corrélatIon entre le rétrécissement (LA/ILA) des voies respiratoIres et ASM. Chez les
controls il y avait 20% de contraction de ASM, mais les poumons sounm: a la MCh
avaient des % de contraction de ASM entre 0 à 80% de la valeur prédite pour des voies
contractées et ceci, pour des voies respiratoires de différentes grandeurs ct
indépendemment de la quantité de ASM.
En résumé, 1) la quantité de ASM ne semble pas déterminer les différences du
niveau de bronchoconst!'ictlOn maximale chez les cobayes normaux; le manque de
corrélation entre les réponses et la quantité de ASM peut-être explIqué par la dl~tnbutlon
hétérogène de la bronchoconstnction auprès des voies respiratOIres étudiées ou encore,
pal' les techniques de provocation; 2) la sensibilité à la Meh semble être reliée aux
différences de quantité de ASM dans les voies aériennes cartIlagmeuses du parenchyme;
3) la variabilité de EC50 reflète possiblement les différences d'aires de surface des
sections transversales des voies respiratOIres; 4) la résistance Dulmonaire ~emble être un
bon indice du niveau de contraction des voies aériennes pUlSqL.' elle démontrait une bonne
correlation avec les paramètres mophométriques calculés ; 5) 1 'hétérogénéité du
rétrécissement des voies aériennes ne semble pas être derminée par des différences de
ASM.
ACKNOWLEDGEMENT
1 would like to thank Dr. James Martin fcr his guidance and support throughout my
graduate studles. Dr. Manin helpcd me develop analytical skills in understanding and
conducting research. 1 admire the patience he has shown throughout both the
development of my experiments and the writing of this thesis. 1 appreciate his support
and encouragement.
1 would also like to thank Tao Du who introduced me to the techniques of
morphometry, and Dr. Nai-San Wang who taught me aboUt the pathology of the lung.
1 am grateful to Mr. Serge Filiantrault and MT. Robert Thomson for thelr valuable
technical assIstance, and to Dr. H. Ghezzo who helped me with the analysis of the data
and provided his expert opinion on statistics. Special thanks to Anne-Marie Lauzon,
AlaIn Gauthier and Nancy MacGregor for their help in editing this thesis.
Finally, 1 thank my friend and colleague Noor-Jehar Kabani for her constant support
and advise. 1 thank my sister Aurora for the many hours she spent performing sorne of
the morphometncal analysis, and most important for her love and encouragement that
made the completion of this work possible. 1 thank my parents for everything.
v
To my family
ABSTRACI RESUME ACKNOWLEDGEMENTS TABLE OF CONTEI-JTS LIST OF FIGURES LIST OF TA BLES LIST OF ABBREVIA';"rONS PREFACE
TABLE OF CONTENTS
CHAPTER INTRODUCTION
1.1 Histoncai Perspective and Definitions
1. ~ Structural Considerations
I.~. 1 Smooth Muscle Distribution
1 ~ 2 The Role of the Epithelium
1 ~ 2a The Epithelial Bamer
1 ~. 2b The EPltlwlium as Metabohc Tissue
Page
III
V
VI
ix XI
Xll
XlI1
1
3
3
4
4
3 ~kchal1lcal Determinants of Airway Responslveness 5
1 3.1 Length-Tenslon Charactenstlc
1.3 2 Load~ acting Ofl ASM
1 J.3 The ProportIOn of ASM in The Airway CIrcll.nference
1 3..+ The Amount of ASM
1 4 Autonomlc Factors as Determinants of ASM rC~p0l1S1 vene~s
10
Il
13
13
15
lA. 1 Structure of the Autonomie Nervous System 15
1.4.2 The Sympatheuc Nervous System (SNS) as Determlllant of ASM Responslven~ss 16
IA.23 Structure and functlon of the SNS
l.·~ 2b V;lnablhty In Adrenergic InnervatlOn
l..f. 2c The Role of CIrculat1l1g Catecholamines
1.-i.3 The Para~ympathetic Nervous System (PNS)
16
16
17
é4~ Determlllant of ASM Rcsponsiveness 18
lA 3b Variablhty 111 Parasympathettc Innervation 19
vi
1 AA The Non-adrenergic Nervous System (NANC) a~ DetermInant of ASM Responsivencss 20
lA 4a Structure of NANC 20
lAAb Phy~iologl'; Role of NANC 21
1 -lAc The Role of NANC m ASM Responsl'v'cness Yl
1 5 r\ero~ol Deposltlon as a Factor ln Responslvenc~s 2-l
16 QuantltatlOn of Alrway Smooth ~lusck 27
1 6 1 ~Iorphometry: Method and l'îKory ), ~I
1.6.2 ~Iorphometry Applted to the Study of Structure and Alrway ResjXl(1~i'lcnt'ss 29
CHAPTER " ~IETHODS 32 -2.1 Aleway Responslvel1e~s to Methadll)IlI\e '1 .L
2 1 1 :\n IIllal PreparatIOn 32 ., 1 Î Plethysl110graphlc Measurements 33 -2.1 ~ l\kthacholmc ProvocatIon TesLIng \6 -'
2. 1 -l Experimental Protocol J7
2.1.5 Hi~tolùglc and Morphometnc Studlcs 17 ., .,
Qllld-frcczlI1g of Gumea Pig Lung~ IX
2 2 1 Animai PreparatIOn JX .., .., .,
['I1<.?r1 mental Protocol \li
~ 2 3 HI<,tologlc and ~lorphometnc StlldlC\ 19 ., , - .' Freqllency-re~ponse Charactenstlcs of the Volume and
Esophageal Pre~~ure Measurcrncnt System~ 40
2A Data Collection ~I
2.5 Data AnalY'iis 42
2.5 1 Physlologlcal Measuremenls ·t2
Î - " - ).- Morphometry 43
2 5.3 Statistical Analysis 45
CHAPTER .3 RESULTS: I. Methachohne Provocation Test 46
3.1 Ph ySlOloglcal Measurements 46
"'ll- ~ Î -' ...... Morphomelry 49 '''./
3.3 DistributIOn of Alrway Size fur Alrways Samplcd VII
S6
ft
3.4 Measurement of Airway Smooth Muscle 61
3.4.1 ASM as a FunctlOn of Airway Size 61
3 4.2 Variability in Airway Smooth N:uscle Among Guinea PigS 67
3.4.3 Variabllity in Alrway Smooth Muscle Among Different Slzed Airways 67
3.5 CorrelatlOns Between ASM and Rrnax and logECso 70
3 5. 1 Intraparenchymal Airways 70
3.5.2 Extraparenchymal Airways
Il QUtck-freezmg of Guinea Pig Lungs
73
80
3.6 Mcthacholtne (MCh) Challenge 80
J.7 Morphometry 80
J 8 Dlstnbution of Alrways Sampled 84
J.Y Alrway Narrowmg 88
3.9.1 DlstnbutlOn of Airway Narrowing Among Ali sized Alrways 88
3.9:2 Distribution of Airway Narrowing in Small, MedIum, Large, and Extra-Large Airways 90
3 9.3 DlstnbutlOn of Alrway Narrowmg In Small and Large Airways According to the Median Airway 92
3.9 -+ CumulatIve Frequency Distnbutions 96
3 10 Dlstnbution of Alrway Smooth Muscle 100
3.11 Alrway Narrowmg as a FunctlOTl of ASM 106
3 12 Smooth Muscle Shortening (SM Shortening) 111
3.12. 1 DIstribution of Smooth Muscle Shortening III
112.2 SM Shortenmg as a Functlon of ASM III
CHAPTER -! DISCUSSION
4. 1 Methachohne (MCh) Responsiveness
4.~ VariabIilty in Airway Smooth Muscle
4.3 Variabihty 10 Alrway Narrowing
BIBUOGRAPHY
vÏJi
116
116
121
125
128
FIGURE
2
3
LIST OF FIGURES
DE:~CRIPTION
Theoretlcal cOllcclltralion-rcsponsc curves.
Mechallicai dctcrminants of airway responsivenc55.
Experlmcntal 'iet-up.
METHACHOLINE PROVOCATION TEST
4 Rcprc~elltatl vc conccntration-response curvc.
51\,B Photomlcrograpll of [:lllnea pig alrway and digitizcd tracing.
6A,B Phot0J1lIcrograpll of airway from guinca plgs #1 and IIJ.
7A,B,C PhotOll1lcrographs of alrways from gumca plg #5.
8 DI\tnbutlon or illtraparcnchymal alrways sampled.
9 FrcqLH:llcy di'itnbution of alrways accordll1g to alrway SiIC.
10 J)\<,tnhu!\on of éurway 5\1100th mu~clc (ASM) among anllnals.
1 1 J)1~tnbllt\DI1 of ;\Sr."l among different sized airways.
1~1\,B 'l'Ile a[)..,olllte and relative vanabiltty of ASM.
CORRELATIONS
13A RlllélX alld /\SM 111 intra- and extraparenchymal airways.
1313 logEC'1I and 1\5M in intra- and extraparenchymal airways.
14/\ Rllld\ and /\SM III 311lall and large intraparcnchymal airways.
14B logEC,o and 1\SM ~l11all and large lI1traparenchymal alrways.
15/\ Rma>, and /\SM 111 mcmbranous and carlilagl110us alrways.
15B logEC'lI and ASM in mcmbranolls and cartilaginous mrways.
QUICK-FREEZING OF GUINEA PIG LUNGS
PAGE
8
1)
35
48
51-52
53
54-55
57
58
08
69
71-72
74
75
76
77
78
79
16A-0 Pllotonl1(:rographs of airways l'rom quick-frozen lungs. 82-83
IX
FIGURE DESCRIPTION PAGE
17 Distnbutlon of airways sampled (guinea pigs #14-19). 85
18 Frcquency distribution according airway size. 87
19 DI'itribution of alrway narrowing versus length of epithelium. 89
20 Airway narrowing (LA/fLA) in small and large airways. 93
21A,B,C Cumulative frequcncy distributions of airway narrowing. 97-99
221\ The I11cdlan LAIILA versus degrce of bronchoconstriction. 101
2213 The I11cclian LAIILA versus the absolute lung resistance. 102
23A,B The rncan LAIILA versus degree of bronchoconstriction. 103-104
241\ Dlstnbution of ASM (non-standardized). 105
24/3 The relative vanability of ASM. 107
24C Distribution of ASM among guinea pigs. 109
25 Alrway narrowing versus ASM. 110
26 Slllooth muscle shortening versus Jength of epithelium. 112
27 Smooth muscle shortening versus ASM. 113
x
LIST OF TABLES
TABLE PAGE
1 Blood gases ]4
2 lung rcsistance in rc~ponsc to methacholine for complete conccntration-response curves 47
3 Weight, Rmax and sensitlvity challenged with methacholine 50
4 Mean size of airways in formahn-lÏxed lungs based on the length of the basement membrane 60
5 A mount of ai rway smooth lI1uscle in ail airways 62
6 ASM in intraparenchymal airways divided accordlllg to the median airway 6J
7 Amollllt of ASM in small, medium and large intraparenchymal mrways 64
8 Amollllt of ASM III intraparenchymal airway~ dlvidcd accordll1g to the presence of cartilage 65
9 ASM 111 the top 25 % of intraparenchymal airways 66
10 lung rc),i'itancc in qUIck-froLCIl Illngs 81
Il Mean size of aIl intraparenchymal alrways in quick-froi'cn lungs bascd on IBM 86
12 Distribution of L I\/ILA in different sized alrways of quick-flOzen Illngs 91
13 Distribution of lAIILA in different sized airways of quick-frozen lungs accordlllg to median airway 94
14 Median value and range of LA/IlA in qllick-frozcn lungs 95
15 Amollllt of ASM in qllick frozen Illngs divided according to meclian airway 108
XI
-
, .. LIST OF ABBREVIATIONS
ASM AW BM EP ECso
OP ILA LA LBM LEP MCh Pes Ptr PTP
SM Raw RI. Rmax VT • V
Airway Smooth Muscle Airway Wall Basement Membrane Epithelium Effective Concentration of drug at which 50% of maximal response is achieved Guinea Pig Ideal Luminal Area Luminal Area Length of Basement Membrane Length of Epithelium Methacholine Esophageal Pressure Tracheal Pressure Transpulmonary Pressure Smooth Muscle Airway Resistance Lung Resistance Maximal Resistance Tidal Volume Flow
xii
1 PREFACE
This thesis examines the variability in the amount of alrway smooth muscle In the
guinea pig and the relationship, if any, to the degree of airway responsiveness observed
in this species. This question has been approached by first performing in vivo
measurements of airway responsiveness to the agonist methacholine, and then relating
them to morphometrical measurements of airway smooth muscle and olher airway
dimensions.
The tracheobronchial tree c,f different animaIs exhiblt di fferences In the degrce to
which constituent airways narrow in response to bronchoconstrictive stimuli. The smooth
muscle in the airways is believed to play Q major role in determining airway narrowing
by its ability to contract in response to a variety of stimuh, and to relax to its initial
length in a reversible fashion. It is possible that differences in airway smooth muscle
may be responsibJe for this variabiJity in airway responsiveness.
So far, several studies have attempted to study the properties of smooth muscle in
vitro, which, faiJing to correlate with in vivo measurements, have led mvestIgators to
conclude that the variation of smooth muscle in response to an agomst IS not due to the
intrinsic sensitivity of the smooth muscle. Attempts to relate the quantity of airway
smooth muscle to a1Tway responsiveness in vivo have not found a relallOnship bul have
been performed on subjects whose lungs were not normal.
The present study attempts to show that the smooth muscle has an important role in
determining variations in airway responsiveness by systematically examining in vivo
xiii
concentration re!'ponse curves among animais of the same species and attempting to
correJate such functionaJ measurements with structural tindings from morphometrical
anaJysis of airways from the entire Jung of these animals.
Four chapters are presented in this thesis. The tirst provides a historical review of
the Iiterature and background information inc!uding structural and functional
considerations pertinent to this thesis. Chapters two an three are divided into two parts,
one dealing with physiological experiments, an the other with morphometry. These two
parts are integrated in the discussion and summary.
xiv
Chapter 1
INTRODUCTION
1.1 Historical Perspective and Definitions.
The pnmary functlon of the re~plratory system IS to transport .ur fwm thl:
atmosphere to the alveolar surface, where gas exchange takcs place. In orùer to carry
this out efficiently, the respiratory system must have a durable structure and a defcnsc
system that will protect it against environmental insults. The upper alrways (nasal
passages, nasopharynx), the trachea, bronchi and non-alveolated bronchioles arl: dCSlglll'd
for conduction while the alv~oli functlOn in gas exchange; betwccn thcsc, a tran'\ltlonal
zone performs both conductIon and resplratory cxchange. Large parl1culatc matcnal IS
removed within the upper airway by impactlon on the mucus blanket and IS o.,wallowl!d.
The lung also has the appropnate mechanisms to remove smallcr respirable partlcJc'\. The
airways 'varm and humidify the aIr as it enters the lung and allow the productIOn of
speech as it passes the vocal cords. The structure of the airway consi~ls of an cpithdial
lining, smooth muscle, cartilage, blood vessels and supporting connectivc tissue. The
airways are therefore not passive conduits since the smooth muscle can contract or rclax
to change resistance to air tlow and regulate ventilatory dead space (62).
The ability of airway smoolh muscle (ASM) to conlract has bccn 'ihown 10 he an
inherent property of s.l1ooth muscle among both humans and thc variolls mélllllnallan
species studied so far (10,32,49,119,120,144,164,183). Thl~ capaclly of thl! ~Illo()lh
muscle 10 contract in response to a variety of stimulI (drug~, chemical irntants,
L.....-_____________ ~ ___ -
osmolaril.y and cold air) is known as non-specific (non-allergie) airway respcnsiveness
(166).
Airway responslveness exhibits a log-normal distnbution and shows a wide rar.ge
bctween hypo- and hyperresponsive individuals. In human populations those individuals
who arc atopic or aslhmatle are likely to be at one end of a eontmuous spectrum of
rcsponsivcncss, while mdlviduals who are hyporesponslve are found at the olher end.
Onc large sludy has looked at the dlstnbution of airway responsiveness to histamine in
a group of 300 randomly sclectcd subjeets (32). This study showed a log-normal
distribution evcn lhough asthmatics and subjeclS wilh allergie rhmitis were II1cluded.
Douglas ct al. (49) dcscribed histamme responsiveness in guinea pigs with ncarly a 100-
fold range between the most and least sensitive animaIs. Snapper et al. (164)
dcmonslrated a similar variabllity in dogs where there was a greater th an 40 fold
dIl1crcnce 111 lhe range of sensiLivIty, and Bai et al. showed this variabIlity in
rcsponslvencs~ in cats (10).
Exact!y what determines the wlde variability III airway smooth muscle responsiveness
III a given populatIOn is not clear. Recent evidence suggests that mechanical factors are
Important. This eVldence IS the subJecl of a recent reVlew by Moreno et al. (124). The
purpose of thls thesis IS LO study one of the possible mechanical causes of this variability,
namely the quanlily of airway smooth muscle.
The IIltroductlOn foc uses on 1) the structural properties of the airways; 2) meehankal
determmants of ASM responsiveness; 3) autonomie factors; 4) aerosol deposition; and
5) quantitation of ASM.
( ,
2
li,
1.2 Structural Considerations.
1.2.1 Smooth muscle distribution.
Smooth muscle can be found from the trachca to the alveolar duets (188). In the
trachea, bundles of smooth muscle fibers are arranged transversely. and attach to the
posterior aspects of the ends of the cartilage nngs. Aiso 111 the traehca. cartilage IS
arranged as a series of horseshoe-shaped rings wllh the open ends locatcd postenor! y
ln the aIrways where carttlaginous plates occupy the cnlire circumferencc. thc 1l111\l'k
coat is arranbed as a i_'yer IIlternal to IL ln faet, the smooth muscle becoll1es oh Il li lId y
oriented and forms irregular spirals down the airway (122,188). This arrangcment is
evident in airway cross sections where the smooth muscle does not appcar to he
continuous throughout the airway perimeter.
Toward more distal airways the cartilage decreases in size and disappears al the levc\
of bronchIoles. As alveoh II1crease in numbcr, the epIlhelium becolllcs scanly, the
submucosa disappears, smooth muscle bundles (as weil as elastic tl~SUC libers) COlltlllllC
splralltng down the alrway and around alveolar mouths (62) Contractlle cclb and
bundles of microfilaments similar to those of srnooth muscle can be round 111 thc alvcolar
interstÎtlUm (62). Because of the dlfferences in SM dlstnbutlon througholll the
tracheobronchial tree, the response to agofllst may be different l'rom sl11all and large
aIrways (89,168).
1.2.2 The role of the epithelium (EP).
The epithelium is potentially an important determinant of airway rcsponsivencss in
3
lt~ role a~ a physlcal barner and as a metabolic tissue.
1.2.2a The epithelial barrier. The epithelium is a physlcal barrier that protects the
airways against temperature and humidity changes, irritants, and forelgn proteins. The
Et> IS Impcrm~ablc to most substances (62). In the tracheal and proximal bronchial
~pllhchum, cihatcd ccll~ extcnd from the lumen to the basement membrane, and attach
to one another at the apex by tighlJunctlOns. Undcr sorne clrcumstances thlS barrier may
be dlsruptcd, for cxample, an antigen can traverse the epii:helium by activatlllg sU(lcrficial
l11ast eclls that WIll open tight junctions by secreting medIators. This permits the antigen
to reach the deeper SM and the mast cells adjacent to it , leading to further mast cell
degranulatlon and releasc of medlators such as histamme, j,eukotrienes, prostaglandins,
and thromboxanes (21). In addition to exposing mast ce])s, disruption of the EP can
expose the afferent sensory fibers of intraepithelial nerves which give rise to the irritant
rcceptor response (130).
1.2.2b The epithelium as metabolic tissue. The epithelium has a function in
metabohzmg substances that participate in the inllammatory response elicited by lung
in jury . Various studies have shown that lung injury can stimulate the EP to produce a
potent chemotactlc factor leukotriene B4 (79,105,136,146), which can contract ASM in
~OI11C SpCCICS. In Vitro expenments of guinea pig trachea show that this factor is 10 times
as potent as histamine in contracting ASM (104). Leukotriene 84 has also been shown
to IOduce airway hyperresponsiveness in dogs (80,135). In addition to producing
4
-
bronchoconstricuve substances, the EP can also producc a SM rdaxlng factor (51)) Whll'll
can inhibit bronchlal SM tone. From 111 vitro expcrimcnts Flavahan ct al. (54) obscrwd
that removal of the EP from the bronchlal alrways of mongrd dogs resultcd ln an
increase in the sensitivity of smooth muscle to scrotomn, accty!chollllc. and IlI'~tal11lnc
Prostaglandins are prod.lced by the eplthelium and bronchoacllve peptides may he
degraded by endopepttdase enzymes located on the clhatcd cplthcllllll1 (16H).
1.3 Mechanical determinants of airway responsivcness.
The mechanical delermmants of alrway responslvcness arc best understood rwm an
analysls of the dose-rcsponse curvc to a bronchoconstnctlvc substance, wllerc the
"respome" provldes an IIldlreet measurell1cnt of alrway narrowlIlg. ProvocaltOIl te,t,
wlth Illstamine have been most frequently uscd to provlde quantitative e.,tilllate~ of ~o
called non-specIfie bronchial reactivity since they were tntroduccd by Curry 111 1946 (37).
Cockcroft et al. (33) standardized these t~sts by look1l1g al the cffects or IIlhalcd
histamine in normal subjects, weil conlrolled asthn"'lics, and patlcnts wlth rhlllllls or
chronic cough. Testlng for non-specifie ain\lay rc~ponsivcnc~~ I~ dom: today hy
performing a dose (or concentratIOn) response cllrve to Inhaled phannacologlc agent,
such as hIstamine or methachohne. Airway narrowll1g IS e~t1Jnatcd frolll change) 111
airway resistance (Raw), or the maximal expHatory tlow (FEYt), or the maximal
expiratory t10w volume curve ('Vmax). The do!:.e-responsc curvc lS lIsually é:lnaly/:co by
measunng the concentration or dose that produccs an arbttranly dclincd rc~p()n~c slIch
as a faIl in FEYl of 20%, or pe20 (33) or PD20 (27); the reactivity, or ~Iope of the
5
dose-respon~e eurve IS less often used.
Several mvestlgator~ (119,174,193) have demonstrated complete concentratlOn
response curves in humans and m patients with mild asthma, when exposed to histamine
(119,193) and methacholme (174) aerosols. Mild asthmaties tend to have increascd
maximal responses (119,193). Subjects with more severe asthma do not appear to reach
a plateau because submaximal doses produce large decreases in FEV! precludmg further
doses (119,193). The presence of a plateau on the dose-response curve indlcates tllat
ASM IS able to shorten maximally ln VIVO withoui causing complete alrway closure.
Macklem (111) and Woolcock et al. (193) have suggested that inereased bronchial
rcactlvlty In asthmatlcs must be eaused by the deticiency of an inhibitory factor that
normally hmlts smooth muscle shorteP.1ng.
Complete concentratlOn-response curves in vivo can be compared to pharmacologie
dose-response curves of alrway smooth muscle in vitro. In c1assical model') of drug
receptor mteraction two factors are usually considered for companng dose-response
eurves: 1) the maximal response (I.e. the plateau), and 2) the effective concentratIon that
produces 50% of the maxImum response (i.e. the EC50). A complete concentration
responso! curve IS one where increasing concentrations or doses of agonist are
admll1lstered untll a plateau develops ln the response. For the purpose of this thesis, the
position of the curve wlth respect to the x-axis will retlect aIrway sensltivity, and the
plateau on the y-axIs WIll retlect the maximal responslveness or maximal alrway
narrowlng. Both of these parameters are Important to consIder separately in describing
the alrways response to agoni st. For instance, figure 1 shows three types of
6
..
".
concentratlOn-response curves that may be encountercd. curvcs wIth thc salllc ma '\ll11al
response but different EC,o. Cl.Jrvcs with the saille EC\o but diffcrcnl rnaxllllai r~~pon'ie.
and curves with dlfferent EC5U and different maximal responsc.
In a theoretical paper Morcno and colleagues (124) dlscusscd the Illcchalllcai
determinants of airway responsivcness and hypotheslzed that Ille factors that n:gulatc the
sensitivlly and the maxImal respol~~e. Thc authors dcscnbc thc vanou'i ~ters whlch kali
to airway narrowmg followmg mhalatlon of a pharmacologlc ago\1l~l. Accordlng tn thcir
schcme (reproduced ln fig. 2), an agc!'lst tïn~t actlvates smooth musdc, causlIlg, Il 10
shorten. The degree of slllooth muscle shortemng IS determmed by the dosc-re~p()me
curve of the muscle, the length-tension characteristics of ASM. and the rclatlOIl'ihlp
between quantlty of ASM and the load that the ASM must oycrcomc dunng shortellll1g.
The overall effect of these events on the external dlamctcr and the éllrway re\l'itallcc
depends on several factors. These mclude the proportIon of smooth muscle round 111 the
clrcumference of the airway, the thlckne<;s of the alrway wall, and the presence of
luminal secretIOns, as weil as the smooth muscle mechamcal advantagc. In thclr analy'il~
of the dose-response curve, Moreno et al. attcmpl to explaln the vanablhty ob~crvcd III
the response of ASM to agomst. The authors argue that an Increa<;c ln ~cn\ltIYlly cou Id
be due to altered depo~llIon of acrosol, an IIlcn:a~c III cpllhcltal pcrmcabtllty, a Ùl:cn:a\c
in the removal of agon\st, or an increase 111 muscle cell to cell coupltng. In contra~t, an
mcreased maxImal response could result from an mcrease 111 smooth mu~clc \trcngth
from an Increase ln the amount of smooth muscle or an altcrcd Icngth-tcn~l()n
relationship, a decrease in the smooth muscle load, an incrcase in wall thicknc~s, or an
7
IIIQ en
f CI) IIIQ &If
, !
A B C
100
r 50
LOG CONCENTRATION OF AGONIST
Fig. 1 Theoretical concentration-response curves:
A) Tvo curves vith the same maximal response but different ECSO.
B) Tvo curves vith thp. same ECSO but different maximal respons~.
C) Tvo curves vith different ECSO and different maximal response.
x = ECSO o = Maximal response
8
1
".'
•
~
STIMULUS
! •. ------~I Agonist-Receptor interaction
ASM ACTIV A-:;:'ON ]
1 Length-Tension Load .. ------..... 4mount of ASl\1
ASM SHORTENlNG
l • EXTERNAL DIAMETER
1 • AIRWAY LUMEN
1
Contractllity
Proportion of muscle in the airway circumCerence
Mechanical Advantage
Wall thickness Secretions
! +--------tl Flow Regime •
t hw
Fig. 2 Mechanical detenninants of airway responsiveness (Reproduced from Moreno et al., 1986)
9
(
Increase in secretions ln the airway lumen.
1.3.1 Length-tension characteristics. Smooth muscle has a length-tension relationship
similar to skelctal muscle (171). Isometric length-tension curves obtained from in vitro
studies of canine tracheal SM (171-173) show that as the active tension developer. during
electrical stimulation increases with muscle length until a maximum is reached at a length
known as Lmax, and then decreases with further stretching. Smooth muscle is able to
develop tensIOn over a greater range of lengths than skeletal muscle, due to its loose
organlzation of sarcomeres and the absence of Z bands to limit the sliding of myosin
filaments. Canine tracheal smooth muscle, for instance, has been shown to develop
active tension when the length is between 10 and 20% of Lmax, in contrast to skeletal
muscle that can contract to approximately 20% of Lmax (171). If ASM is at a length
shorter or longer than the optimallength, th en shortening will be submaximal for a given
degree of activation. Thus the initial length, in relation to Lmax, will determine the
extent to which ASM will shorten, and differences in ASM length along the length
tension curve may be expected to result in changes in the maximal "esponse to an
agonist. An altered length-tension relatlOnship, that is, a change in the ability to generate
tensIOn in the maximal response in VIVO to a maximal stimulus could potentially account
for differences in the position of the dose-response curve in relation to the y-axis.
The smooth muscle length in vivo is presumably determined by the preload on ASM.
However, little is known about the operating length of ASM in vivo. Sorne studies
(69,124) have suggested that bronchial SM is near Lmax at physiologie transmural
10
pressure. Even though ASM can shorten to about 20% of ItS initia! Icngth in vitro. this
does not happen in vivo. An altcred length tensIOn relationship cou Id account for the
apparent discrepancy. If in vivo ASM were not at Lmax, maximal stllllulatlon would
produce less than 80% shortening (124).
1.3.2 Loads acting on ASM. Decreased load on ASM may reslilt in an inCfeased
maximal response. The loads that aet on ASM are both viseolls and clastic gcncratcd by
structural elements within the airway itself and by sllrrounding tissue, as weil as by the
tension produced by the transmural pressure. In the large extraparcnchymal airways the
major load is provided by the elastic recoil of the cartilage rings. In intraparcnchymal
airways the load is provided by the tethering effeci of the lung parcnchyma on éllrway
walls. The elastic recoil caused by cartilaginous rings in the trachca and the transmural
pressure generated by the tethering effect of lung parenchyma on intraparcnchymal
airways provide the preload to stretch ASM 10 Lmax, and an aftcrload that may IJmlt
ASM shortemng. ThIs would mean that a breakdown III the IIlterdcpcndcncc bctwccn
parenchyma and airway wall, as suggested by Macklem (111), could allow unimpcdcd
shortening to occur and result in increased responsiveness.
The role of cartilage was studied by Dekock (42) who observcd that .vhen the
posterior membranous trachea was incised, cartilage rings sprang outward to a positIOn
with a greater radius of curvature. Thus when the cartIlage IS movcd away l'rom its
resting position, it generates an elastic load that opposes ASM shortcning during
contraction. Without the contribution of this load to limit maximal SM shortcning the
II
ASM could continue to shorten until the airways were compietely closed. This notion
is supportcd hy studies done by Moreno et al. (123,125,126) where rabbits were injected
with intravenous papain to soften the cartilage. Their results showed an increase in the
maximal SM shortenmg JO VItro and in the maximal airway narrowing in vivo. The same
was found !n a morphometnc study done in pigs where cutting the tracheal cartilaginous
rings resulted in an mcrease of ASM shortening (93). Furthermore, McCormack and
colleagues (117) found that there was an increase in the baseline airway resistance as a
result of the softening of the cartilage (117). These observations indicate that cartilage
plays a role in i) regulating baseline airway caliber, by presenting a preload to ASM to
coumeract baselme ASM tone, and ji) limiting ASM shortening, since softening allowed
excessive ASM shortening.
The relevance of the elastic load of lung el as tic recoil in determining airway
narrow~ng was shown by Ding et al. (45) who changed lung volume to alter the load on
ASM. They challenged normal subjects with Meh and constructed a dose-response curvc
lIslng mcasurements of pulmonary resistance (Rd during tidal breathing at functional
rcsidual capacity (FRC) and at Jung volumes above and below FRe. They found that
whereas changes in lung volume had slight effects on pulmonary resistance at baseline,
lung volume caused major changes in airway narrowing following MCh. Above FRe
there was significantly less airway narrowing and below FRC there was more airway
narrowmg. The changes 111 lung volume resulted in changes in aIrway narrowing al high
doses of MCn and 10 maximal airway narrowing. The authors interpreted their findings
as indlcating that changes in lung volume alter the forces of interdependence between the
( . 12
airways and the parenchyma that oppose ASM contraction.
1.3.3 The proportion of ASM in the airway circumference (PMe). A larger PMe
could increase responsiveness (124). ln the trachea, for Instance, about 20% of the
circumference is occupied by muscle, whereas in the peripheral alrways the sllloolh
muscle completely surrounds them, probably in a spIral fashlOn railler than
circumferentially (see section 2.0). In the bronchi, the smooth muscle arrangement is
somewhat intermediate between the trachea and the peripherat airways. The proportion
of muscle in the circumference of the alrways could vary among rndivlduals and thal
could determine differenr.:es in maximal narrowing. As dlscussed by Moreno ct al. (124)
the externat diameter of an alrway decreases hnearly as the PMe increascs for a glven
degree of ASM shortening. Consequently an alrway wlth higher PMe will have grcatcr
narrowing for a glven amount of ASM shortenlllg. Differences III alrway gcolllclry of
the central alrways, for lIlstance, could be illlportant sJnce they contnbutc a large
proportion to airway resistance and are the major sitc of tlow hlmtation (112,113).
1.3.4 The am ou nt of ASM. An increase in the amount or strength of ASM may resull
in increased responsiveness. Schellenberg et al.(158), DeJongste et al.(40) and Bal (8)
have shown that ASM from subjects with asthma (8, 158) or obstructIve pulmonary
disease (40) who have bronchial hyperresponsiveness In vivo, has increascd Mrcngth,
measured by isometric force developmcnt, in Vitro. It is not c1ear If an incrcasc in
strength is associated wlth an increase in the amount of airway smooth musclc. Annour
13
ct al. (6) studied the relationship between bronchial hyperresponsiveness to methacholine
and ASM structure and found a significant correlation between the maximum tension
change In response to histamme and the volume of SM measured. The same study,
howcvcr, faIlcd to show a rclatlonship bctween maximum tension to carbachol and the
volume of SM measured, and found no correlation between in vivo responsiveness to
methacholine and in vitro sensltivity to carbachol or histamine. Other studies that have
attempted to relate in vivo and in vitro responses have not shown a relationship
(7,20,41,! 82,185). The major problem with these studies, however, is that they have
been performed on subjects whose lungs were not normal. In addition, the tissues
resected were taken from one part of the Iung and are not Iikely to be representative of
the whole organ. So it is conceivable that a relationship exists between in vivo
responsiveness and the amount of ASM. A recent study by Nagai et al.(l34), found an
Increase In the smooth muscle and other morphologie changes in the airways of guinea
pigs in whlch recurrent asthma-like breathing was induced by acetylcholine rxposure.
ln another study, Sapienza et al. (157) showed that ovalbumin challenged animaIs had
a 2-fold increase in airway smooth muscle compared to controIs. The findings that the
acetylcholine and ovalbumin-exposed animais had more ASM than controIs suggests that
the amount of ASM may be capable of growth. Such plasticity could be important in the
context of disease.
14
.'
1.4 Autonomie faetm's as determinants of airway smooth musch.' .. rsptlllsh·l'IIC.'~S,
It has been suggested that differences III airway responslvcncss could b~ call~ed hv
an imbalance between the cholmerglc and sympathetIc ncrvous systems. For c:\amplc.
responsiveness could be due to exaggcratcd actIOn or lI1ncrvatlOn of thc chollllcrgic
(147,161), alpha-adrenerglc (147,178), and non-cholinergie cxcitatory (109) brandIes of
the autonomie nervous system. Similarly, excessive alrway rcsponslvcne~s rould he the
result of decreased beta-adrenergic (147,178), or decreased non-adrcllcrglc inlllbitory
(VIP) nervous activlty (13,116,150).
1.4.1 Str'ucture of the Autonomie Nervous System.
The ANS consists of the sympathetic, parasympathetic and non-adrenergic nerVOll'i
systems. Each branch has a potential role ln altering nervous lIlput 10 the mrway smooth
muscle (ASM) and in modifying Its contractIle response. The general pattern or
mn2rvatIOn is as follows. The vagus nerves and fibers from the uppcr four to live
thoracic sympathetic gangha join at the hllum to form antenor and postcnor plex lIse'i;
from these the two malll groups of nerves give nsc to the peribronclllai and penartcnal
plexuses. The peribronchial plexus further dlvides into an extrabronchlal branch that Ilcs
outside the cartilage, and a subchondrial branch that lies bctwecn the cpithclllllll anJ the
cartilage. The a1rway~ l'rom the trachea down to the re~plratory bronchiolc~ arc ~lIppllCd
by nerves (thls II1clude~ ~lIbl1lllCOsal glands and bronclllal vc~~cls) (59, J03, 169).
The ANS controls vanous aspects of airway function, IIlcluding control of alrway
smooth muscle tone, secretion of mucus from submucosal glands, transport of flllid
15
{
•
acro!.!. airway cplthellum, the release of mediators from mast cells and other
inflammatory cells, and pcrmcabihty and blood flow in the bronchIaI circulation.
1.4.2 The Sympathetic Nervous System (SNS) as a determinant of ASM
responsiveness.
Variability in airway responslveness could result from variations in nervous activity
or in the level of clrculating catecholammes. The SNS consists of sympathetic nerves,
wlllch release norepinephrine near the target organ, and of the adrenal medulla, which
rcleases catecholammes, mostly epmephrine, into the circulation.
1.4.2.a Stmcture and function of the SNS. The sympathetic nerve supply to the 1 ung
ongl11ales from lhe upper SIX thoracic preganglionic fibers that terminale 111 the
cxtrapulmonary stellate gangha. Postganglionic fibers ex tend from here to the lung, and
enter at the hi1um where they join cholinergie fibers to fOTln a dense plexus that
surrounds the airways and blood vessels (14,103,149).
1.4.2.b Variability in adrenel'gir innervation. There is little or no direct adrenergic
Innervation of ASM in most species. Most anatomical studies have demonstrated few
adrcncrgic libers in bronchial SM and none in bronchiolar SM (149,150). Nonetheless
sOllle studlcs have shown variability in adrenergie innervation among different speeies
(73,149). ror instance, in dogs there seems to be some degree of sympathetic tOile.
Thcrc could be differences in the extent to which adrenergie nerves cause bronchodilation
16
. ,-
by inhibition of the parasympathetlc pathways. In support of thls idca IS the tindlllg thal
adrenergic fibers have been demonstrated 111 varying degrees In para~yl11pathdlC .1Ifway
ganglia in humans and in other specles (149). This mcans that l10rcplllcphnnc cOllld
affect transmission of acetylcholine from parasympathctic ganglia (11), and inhlbll rclcasc
of ACh From parasympathetic nerve endings (184) in di fferent degrees. But thls latter
effect is unlikely to be of major consequence since adrenergic mnervation IS seant y or
absent.
1.4.2.c The role of circulating catccholamincs. Even though ASM i~ supplicd by kw
adrenergic fibers, it is relaxed oy adrenerglc agonists. ThiS suggcsts Ihal 5111001h lllll,>clc
tone may be altered by catecholam1l1c~ releascd l'rom the adrenal lIledulla, and thal
impaired release could lead to hyperresponsiveness of the alrways. For cxample '\tllUlI:'i
done in guinea pigs have shown that beta-adrenergic blockade incrcascu IlISlam llll!
induced bronchoconstriction, suggesting a protecti ve effect by circulating catecholam i nes
(50,51). This effect, however, has not been observed in normal hUlTlans. But in
asthmatics, beta-adrenergic blockade results in bronchoconstricllon (118,197); t1m
abnormality ha;; bœn observed during stressful situations such as cxerclsc (12) whcrc
catecholamines fail to rise From baseline concentrations which are otherwisc normal (12).
Yet the fact that in humans adrenalectomy or beta-adrencrglc blockadc do not CélUo,e
hyperresponsiveness, ll1dlcates that a deficit in catecholamines 15 nut a major call~atlvc
factor in asthma .
17
1.4.3 The Parasympathetic Ncrvous System (PNS) as a determinant of ASM
responsi vcncs~.
An mcrcase In parasympathetic activlty could lead to an abnormal contractile
rc~ponse of ASM. Parasympathclic nervous aClivily is responsible for maintaining tone
at rest in the airways of normal humans and animaIs (192). Differences in tone betwecn
differcnt ~pCCICS or among anImaIs of the same species could also account for differenccs
in responsiveness.
1.4.3.a Structure and function of the PNS. The parasympathetic pathways that
regulate the airways are made up of motor fibers and sensory fibers contained in the
vagus nerves.
1) Motor fibers ln the vagus nerves. Vagal nerves extend from the central nervous
system and synapse in parasympathetic ganglia in the airway walls; postganglionic fibers
then cxtcnd ta l11uscarinic receptors on ASM. Stimulation of this pathway results in a
bronchoconstnctton that is potentiated by acetylcholinesterase inhibitors and blocked by
atropine, a muscarinic antagonist that blacks postganglionic cholinergie pathways
(25,35,139). This shows that vagal nerves can cause bronchoconstriction by releasing
acetylcholine from postganglionie nerves.
2) Sensory fibers contained in the vagus nerves. These fibers stem from many sensory
cndings. For example, these extend from the nose or epipharynx, arterial baroreceptors,
or slowly adapting pulmonary fibers; stimulation of these sensory endings leads to a
reduced parasympathetic motor activity and causes reflex bronchodilation. Other sensory
18
.. '
endings are found in the larynx and peripheral airways; stimulation of lh~s~ fÏb~rs
increases parasympathetic motor activity and leads to bronchtl"constnctlOn (22, Dl).
1.4.3.b Variability in pal'asympathetic innerv~ltion. An increasc III parasympathctlc
activity could result in an mcrease in smooth muscle rcsponsivcncss. This hypothl''H';
was tirst studied in patIents with asthma. For instance, various SlllllUh lhal produœ
bronchoconstriction in asthmatics have been shown to stimulate sensory ncrvc activlty 111
the vagus (129). Muscarinic antagonists have been found to be good bronchodilators
(26,29), and muscarinic agonists potent bronchoconstrictors (37). Stlldics by Simonsson
et al. (161) observed parasympathetic involvement in patients who exp~nl'Ilced a
bronchoconstrictor response to histamine aerosol, cold air, cltnc aCld, and rapld
respiratory manellvers, that was decreased by atropine. The incrl'a~cd ~11l0()lh mu~clc
tone found in patients with asthma (36) and chronic bronchitls (98) may be due 10
enhanced vagal activity, or It may be an exaggeraled response to normal vagal tone.
Finally, normal resting lone and mcreases m it may result From sllll1ulalion of ~ensory
nerves due to output From the central nervous system via the vagal cffcrcnl~.
In addition, differences in responsiveness withll1 the same lung, duc to the 1I1lcven
distribution of cholinergie innervation throughout the airways (132), may also account
for sorne of the intersubject variability. Stimulation 0; the vagus has bccn shown to
con strict the large airways but have minor effccts on the small airways. But tlm
distribution of innervation or indeed the muscarinic receptors could vary l'rom ~llbJcct 10
sUbJect and lead to differences. This possible mcchanism docs not appcar to have bccn
19
cxplorcd.
Dcsplte the potential contribution of an abnormality in parasympathetic innervation
to the variability of airway responsiveness, the major determinant is likely to be a post
rcccptor mechanism since smooth muscle contraction can be demonstrated by various
chemically unrelated substances (120, 159). Furthermore, it has been shown by
electrical field stimulation of the Isolated guinea pig trachea that muscle contraction is
enhanced by cholinesterase inhibitors, eliminated by atropine, but is not affected by
hcxamethonium (a ganglionic blocking agent) indicating that the contraction is due to
stimulation of post-ganglionic nerve fibers (60).
1.4.4 The non-adrenergic, non-cholinergie nervous system (NANC) as a determinant
of ASM responsiveness.
ln many species ASM is innervated by an inhibitory nervous system that is neither
adrencrgic nor cholintrgic (13,116,150). Through in vitro studies NANC has been
rccognized as the predominant inhibitory nervous pathway to ASM in humans (13). This
lindmg suggests that an abnormality in this nervous system may cause bronchial
h yperrcsponsi veness.
1.4.4.a Structure of NANC. The anatomie distribution of these nerves to the airways
has not been clearly established. However, nonadrenergic inhibitory nerves have been
dl.!monstrated by in Vitro studles ln several species, including humans (13,116,149).
These ncrvcs have also becn shown III cats (44,88) and guinea plgs (31) by in vivo
20
experiments where the vagus was stimulated after pharmacologie blockad~ of l'holincrgll'
and adrenergic pathways. In additIOn, a non-cholinerglc excltatory effcct has be~11 ... howl1
in vitro 111 gllinea pig bronchi and trachca (5). In hurnans the dClllonstr,lllon of tlw,
pathway has not been consIstent (99,109).
1.4.4.b Physiologie role of NANC. Il has been difliclIlt ta show the fUIlCllOI1 of L1m
nervous system, especially as It relates to ain."ay responslvcncss SIl1CC the ~xact
neurotransmitter of NANC is not known and no speclfic antagonist has been devclopcd.
Electron mlcroscoplc studles have demonstrated "p" type granules, as weil as
cholinergie and adrenerglc vesicles, \11 nerves of human airways (100), mdlcalll1g that
peptides may be the NANC neurotransmitters. Vasoactivc \I1tcstmal peptIde (VIP) and
peptide histidIne isoleucine (PHI) are two peptidc~ wlllch relax ~l1loolh muscle. ;\ large
body of eVldence supports VIP as the neurotransmItter of nonadrcl1crglc Illhlbltory nerve ...
in the airways (13,46,153). VIP was first discovcrcd by Sald as a va~oaClJve sub~tance
mlung extracts (155), and ~ince then VIP has been found in neurons and nerve tertlllnal'i
of ASM of animal and human lungs, near submucosal glands, and \Il bronchlal and
pulmonary vessels (100, 46). Histologlcal studics mdicatc that VIP may reslde 111 the
same nerve terminais as acetylcholine (100). In addition to havmg a direct errect on VIP
receptors of target cells, VIP could affect acetylcholine releasc prejunctlonally, or
cholinergie reeeptors postjunetionally. The NANC system may not bc a distinct neural
pathway but resuIt from concomitant release of peptidergic transmiltcrs and conventlonal
neurotransm i tters.
21
Said et al. (54) studied the function of VIP and showed that VIP can relax s~!ooth
mu~c1c ln VItro, unaffected by adrenergic or cholinergie blockers. In addition, VIP
mimlcs the clectrophyslologlcal response seen in ASM as a result of NANC nerve
~tllnulatlon (25). Studles donc In VIVO In dogs have observed that inhaled VIP protects
against hIstamine mduccd bronchoconstnction (153). ln humans the role of VIP is still
unrcsolvcd. In Isolated human alrways VIP appears to have a relaxant effeet (39) which
has bccn dcscribed as small In one study (39) but about 50 tlmes more potent than
Isoprotercnol 10 another (143). In vivo studies done in humans have shown only a weak
protectlve effeet against histamine induced bronchoeonstriction (127).
Substance P (SP) has been proposed as the neurotransmitter of nonchohnergie
cxcitatory ncrves in the guinea pig from ln vitro studies which have shown a component
of bronchoconstnctlOn that IS not inhlbited by atropine. Even though, 111 vitro, SP
contracts a;cway smooth muscle of various species, including humans (109,142),
II1halauO/I of SP has not bt:en shown to have a signifieant effect on airway functlOn in
asthmatlcs who are hyperresponsive to histamine IOhalation (142). AIso, since in vitro
cxperiments use electncal field stimulatIOn to elicit peptidergic excitatory responses, and
thcse rcsponses result from retrograde action potentials, the physiological role of these
pathways 111 the intact animal is quite uncertain. It is possible that tachykinin release
l'rom C tiber afferents may be of importance as a result of local axon reflexes.
1.4.4.c The role of NANC in ASM responsiveness. It is possible that if the
mcchanism of peptide release in the non-adrenergic inhibitory nerves became defective, :1'
t
22
an exaggerated bronchoconstrictive response could result. If this neural IIllluencc \Vere
significant, interrupting preganglionic connections by parasympathectomy would be
expected to cause a notable increase HI the extent of induced bronchoconstnction. This
suggestion is not supported, however, by experiments donc to study the dfcct of
parasympathectomy on 1I1duce~ Jronchoconstriction in the cat (9,10) which showed that
a NANC neural influence was demonstrable but small. Although rcccl1tly VIP
immunoreactivity has been shown to be absent in asthmatic tissue (138), no dercet in
NANC mechanisms has been found in asthmatlc patients (8, 102).
It IS not clear to what extent SP, as well as other neuropeptides bcllcvcd Lo aet as
neurotransmitters such as calcitonin gene-related peptide or gastrin-rcleasing peptide,
contribute to alrway smooth muscle responsiveness (14).
23
,-
.
1.5 Aerosol deposition as a factor in airway responsiveness.
ln this thcslS bronchoconstnctton was induced in the guinea pig using aerosols of
rnethacholine (Mecholyl or acetyl-b-rnethacholine chloride). This drug is a cholinergic
agent whlch acts as a rnuscarinic agonist on airway smooth muscle. As an acetyl ester
it IS hydrolyzed by acetyl cholinesterase but at a slower rate th an acetylcholine, so its
action IS prolonged.
ln the rnamrnalian respiratory system MCh acts to i) contract smooth muscle, and
Il) increase bronchial secretions (66,. These effects will resuIt in increased airway
resi stance , depending on the sensitivity and responsiveness of the SM a)ong the
tracheobronchla) tree, and on the characteristics of aeroso) deposition. The properties
of smooth muscie have already been discussed (sec sections 1.2, 1.3, 1.4).
Differences in the deposltion of aerosol in different regions of the lung may
account for sorne of the variabililty in the airway responses measured. Deposition of
IIlhaled particles 10 the lung depends on the physical properties of the aerosolized
particles and (ln the physical dimensions of the airways (4,196). For instance, heavier
and larger particles are likely to be deposited in central airways, where they become
impacted by turbulent f1ow. Smaller particles are likely to reach the periphery; here low
tlow rates result in laminar f1ows, and deposition is determined by Brownian motion and
gravitatlOnal sedimentation (175,196).
Commercial nebulizers produce aerosol particles with a wide range of sizes. The
widc1y used Hudson nebulJzer produces particles that range in median mass diameter
(MMD) from 1.2 li to 15 u. In the present study the ultrasonic nebulizer (De Vilbiss)
24
1 was used, which delivers particles with a MMD of 0.5 to 10 u (85 % of the particles arc.!
4.5 to 6.0 u). Maximal peripheral depositlon occurs with partides 2 to 4 li (1 g 1).
Uptake and recirculation of inhaled Meh may contribute to the amount thal rcaches
peripheral airways.
Another factor that may influence aerosol deposltion could be airway narrowing of
proxImal airways, which may happen first and may prevent, to sOl11e extent, the dchvcry
of aerosol partlcles beyond constricted p01l1ts, toward distal airways.
A complete lung model of aerosol deposition in the guinea pig is nol prcsently
available. Most of the models currently studied have been derived from human lungs.
Weibel's symmetric lung model (189) with a dichotomous branchll1g system consistlllg
of 23 generations, has provided a simple way of studying gas diffusion and l'article
depositlOn. But smce the alrways are believed to be asymmelnc, Weibel's ll10dcl may
have overestimated the number of airway structures. The firsl asymmetnc lung model
proposed by Horsefield and Cumming (82) was not sufficiently complete to be lIscd for
a deposition study. With the deveJopment of morphologlcaJ techniques, other asymrnetric
models have been proposed that are compatible with expenmental data (70,139,195).
While these models have been useful for deposition studies, there arc large structural
differences among them that make predictions of aerosol deposition somewhat dlflïcult.
Nonetheless the lung models probably reflect that dlfferenl branching structures occur 10
different subjects. Inter-subject vanabihty of human lung morpholopy could then result
in differences 111 depositlon among individuals under similar brcathing conditIOns. This
could be a confounding variable 111 the variabIlity of responses ob~crvcd in the gUInea plg
25
wh en challenged with methacholine if differences in branc!iillg structure occur in these
animais.
26
1.6 Quantitation of airway smooth muscle.
There are various methods of quantitating ASM. Two of these are i) morphomctry.
and ii) measurement of myoslIl (114). In thls thesis quantitation of ASM was pcrformcd
by morphometry, which is a more standard approach whereas the measllrcment of myosin
as a method of quantitating contractile protein had not been widely applicd.
1.6.1 Morphometry: method and theory.
The paper of Huber and Koessler published in 1922 was one of the tirst studlcs 10
provide detailed measurements of airway smooth muscle (85). These workers analYlcd
histological sections of the human lung with the light microscope. They measlired the
average thickness of the bronchial muscle in millimeters which they then rclatcd to the
outside airway diameter. Even though they reported an increa-,c in ASM in patients wlth
status asthmaticus, various questIOns arose because thl' rcsult could havc bccll due to
contraction of bronchlal 5mooth muscle and not necessarily to hypcrtrophy of Il.
Methods developed later involved measuring the arca of muscle rather th an thlck!lcs~.
Area varies liule with degree of contraction, since the volume of the muscle rcmains the
same. Dunmll (52) was one of the first to use the point-countmg mcthod to cstllllatc area
ratios in the lung. This method had been used in other fields such as geology to
quantitate the composition of rocks. Chayes (30), a geologist, followed the Dclcssc
principle of area to volume proportion and provlded a mathematical proot" that pOllll ~Ulll~
c10sely estimated relative areas. Dunnill and other workers (28,190,191) dcvclopcd thls
technique to assess more accurately the pathological abnormalities in a hum::n organ.
27
Later studies employed the point-counting method to quantitate BSM in asthma,
chronic bronchitis and emphysema (53,74,83,84,110). Thus Jung morphometry has
cmerged from an interest in measuring the structural components of the lung.
Morphometry in its broadest sense, is the measurements of blOlogical structures, by
using microdissection and seriai section reconstruction. It attempts to derive numerical
information from macroscopic or microscopie structures (3). It is useful tool because
It can provide information about a three-dimensional structure from a two-dimensional
image or section. Baslcally it can estimate surface area (or volume) using simple test
grids supenmposed on the structure to be mf"..asured; the procedure is done repeatedly and
randomly so that the estlmates are based on probability.
Sorne useful applications of morphometry are in the correlations of structure and
functlon. But while morphometrical analyses can provide information about three-
dimensional structures from two-dimensional images or sections, care should be taken
when attempting to make correlations with measurements done in 'JVO or in vitro. This
applies in particular to studles invoJving humans where the samples were obtained from
areas next to resections during lobectomy or pneumonectomy. In mauy cases sampI es
may not be representative of the entire organ and even worse they may have been
contaminated by the diseased portion of the lung.
Still, if a correlation between structure and function has not been found, this does not
necessanly rule it out S1I1ce morphometrical methods are not exhaustive but probabilistic.
But even with a representative number of samples the method employed may not be
detecting the real value of the parameter in question. One way to overcome sorne of the
28
ft
sampling problems is by using small animaIs where the entire lung can be sampled. The
present study will measure various parameters. particularly the smooth muscle in the
airways of guinea plgs.
1.6.2 Morphometry applied to the study of structure and airway responsivcncss.
Various investigators have employed morphometry to relate airway responsivcncss
to structural components of the lung. One study attempted to provide an index of
bronchiolar narrowing in disease by measuring size of the smalt alrways (bronchIOles),
in relation to their accompanying artenes in normallungs (19). Yanta ct al. (194) 1001\c<.I
at the epithelial thickness, secretory cell number, SM thickncss, and airway 1ll1lCOllS
gland number and size III responsive and unresponsive dogs, uSlIlg the point-collntlllg
,
t 1, i. ~
method. They found that the r,lore responsive dogs had thinncr cplthclilll11 and hlgher
secretory cell counts than less responsive dogs. But they found no slgnificant dlffcrcnccs
1
r, l
between the two groups with respect to SM thickness or mucous gland number and SIZC.
~t ~' f ~ ?
Sobonya (167) made measurements in lungs of long-standing allergic asthmatics 111
an attempt to characterize structural parameters that would correlate with chronic air-flow ~
t obstruction. The bronchial SM thickness was slgmficantly highcr ln asthmatlcs versus 1
f ~ i' ~
1 r ~
the control and so these two groups could be differentiated using thls parametcr. But the
percent of ASM was not significantly higher tn asthmatics compar{'d to control'),
indicating a proportionate increase in other elements in the alrway wall.
Armour et al. (6) investigated the relatlOnshlp between bronchial hypcrrcsponslvcnc~s
29
l 1':
1
"," ~,
t,
to methacho1ine and airway smooth muscle structure and responsiveness. They ca1culated
,
the proportion of smooth muscle in airway preparations and related changes in
responslveness to methachohne ln vivo or carbachol in vitro to SM hypertrophy or
hyperplasia. Although therc wa~ no correlation between in vivo responsiveness to Meh
and in vitro sensltivity to carbachol or histamine, there was a significant correlation
between maximum tension change in response to histamine and volume of SM measured.
A series of studies performed by James et al. (90,91,93) applied morphometry to
more fully characterize various airway dimensions. They examined histological
preparations using a microscope fitted with a camera lucida; the microscopie image of
the airway was superimposed onto the digitizing board of a microcomputer. Sorne of
their findings provide useful information on airway structure. For instance, they have
shown that the internaI penmeter defined by the epithelial surface is constant whether the
smooth muscle is relaxed or contracted, despite changes in lung volume and SM tone
(90). The same authors also measured the airway wall thickness in guinea pigs acutely
exposed to cigarette smoke but failed to show a correlation to airway responsiveness
(91).
Gther morphometric studies ha' ~ looked specifically at smooth muscle distribution.
Ebina et al. (55) carried out a stuc lof ordinary autopsy lungs and found that the relative
thickness of smooth muscle increased toward the periphery of the lung. Increases in SM
thickness in membranous bronchioles cou Id reflect a direct response to tension and, as
the authors hypothesize, greater airflow regulation in the terminal airways. This
suggestion, however, is not supported by the finding that in sorne asthmatics SM
hypertrophy is found throughout the range of the airways studied (55).
f
30
OBJECTIVES
1) To examine the relationship between the amount of airway smooth muscle and
the airway responsiveness to inhaled aerosolized methacholine (MCh) in guinea
pigs. Complete concentration-respon3e curves will be performed in vivo in
mechanically ventilated euin~~ pigs. The maximal response (Rmax), and the
concentration of MCh required to reach 50% of the maximal response, the ECso,
will then be compared with the amount of airway smooth muscle (ASM)
measured by morphometry.
2) To study if a relationship exits between airway narrowing and the degree of
bronchoconstnction. This will involve characterizing the site and distribution of
bronchoconstriction followmg MCh challenge. Here another group of guinea pigs
is exposed to aerosols of MCh but their lungs are tixed at different Icvcls of
bronchoconstriction. That is each lung will be fixed at 15 %, 40%, 60%, 75 %.
or 100% of Rmax. Airway narrowing will be determined by morphometry by the
ratio of the airway luminal area (LA) to the Ideal luminal area (ILA). The
relationshlp between airway narrowing and airway smooth smooth muscle will
aIso be studied here.
31
r
Chapter 2
METHODS
Two series of experiments were performed on the guinea pig and consisted of the
following: a) Methacholine provocation testing to establish airway responsiveness to
methacholine, and the removal and fixation of the lungs for measurement of airway
smooth muscle. b) Methacholine provocation testing to achieve a desired point along
the concentratlon-response curve, at which time the lungs were quick frozen with liquid
nitrogen.
2.1 Airway responsiveness to methacholine.
2.1.1 Animal Prepal'ation.
Experiments were performed on 13 male Hartley strain guinea pigs ranging in weight
between 290 to 420 g which were purchased from Charles River (St. Constant, Quebec).
Guinea pigs were anaesthetized with uretha."1e (1.5 g/kg i.p.). Supplemental anaesthesia
equivalent to one-third of the initial dose was given when necessary. Rectal temperature
was monitored using an electronic thermometer and body temperature was maintained
using a heating blanket. A tracheostomy was performed for mechanical ventilation. The
ventral surface of the neck was incised longitudinally in the caudal direction from the
level of the thyroid cartilage. The trachea was exposed by dissecting and retracting the
sternothyroid and sternohyoid muscles and a smalt incision was made on the ventral
surface of the trachea between the third and fourth cartilaginous rings. A thin
32
" r
1 polyethylene tube (PE 240, 8 cm long) was passed through the stoma for a distance of
about 1 cm into the tracheal lumen. The catheter was held in place with surgical thread
and cyanoacrylate glue. After tracheostomy the animaIs were paralyzed with
pancuronium bromide (0.1 mg/kg i. v.) and mechanically ventilated (model 683. Harvard
Apparatus, Southnatlck, MA) at a constant tidal volume of 5 ml per kilogral11 and at a
freqvency of 60 bpm (lOG). Supplemental oxygen was given to mamtain artcnal oxygcn
above 100 .11mHg. Arterial blood sample3 were obtained in 3 guinea plgS to detcrminc
blood gases and to confirm the adequacy of ventilation (table 1).
2.1.2 Plethysmographic measurements.
The animais were placed supine inside a constant volume plethysmograph (Fig.3).
The plethysmograph consisted of two cylindrical Plexiglas chambers (3 liters total
volume) interconnected by a Plexiglas tube (3 cm ID, 4.0 cm long), simllar to that
described by Vinegar et al. (186). One chamber was occupied byan animal while the
other was filled with copper mesh to maintain isothermal conditIOns. The pressure ,"side
the box was restored to atmospheric pressure by periodically openmg a sideport. The
plethysmograph was connected ~<J a differential pressure transducer (Vahdyne MP -
45 + 100 cmH20) and calibrated by measuring the change in volume for a given change
in pressure.
Volume was determined from pressure changes in the plethysmograph. Airtlow was
obtained by differentiation of volume. Esophageal pressure (Pes) was measured using
a saline-fi lied polyethylene catheter (PE-90, 30 cm long), placed in the esophagus and
33
polyethylene tube (PE 240, 8 cm long) was passed through the stoma for a distance of
about 1 cm into the tracheallumen. The catheter was held in place with surgical thread
and cyanoacrylate glue. After tracheostomy the animals were paralyzed with
pancuronium bromide (0.1 mg/kg i.v.) and mechanically ventilated (mode1683, Harvard
Apparatus, Southnatick, MA) at a constant tidal volume of 5 ml per ldlogram and at a
frequency of 60 bpm (lOG). Supplemental oxygen was given to maintain arterial oxygen
above 100 mmHg. Arteriat blood samples were obtained in 3 guinea pigs to determine
blood gases and to confirm the adequacy cf ventilation (table 1).
2.1.2 Plethysmographic measurements.
The animaIs were p]aced supine inside a constant volume plethysmograph (Fig.3).
The plethysmograph consisted of two cylindrical Plexiglas chambers (3 liters total
volume) interconnected by a Plexiglas tube (3 cm ID, 4.0 cm long), similar to that
described by Vinegar et al. (l8G). One chamber was occupied by an animal while the
other was filled with coppel" mesh to maintain isothermal conditions. The pressure inside
the box was restored to atmosphenc pressure by periodically opening a sideport. The
plethysmograph was connected to a differential pressure transducer (Validyne MP -
45 + 100 cmH20) and calibrated by measuring the change in volume for a given change
in pressure.
Volume was determined from pressure changes in the plethysmograph. Airflow was
obtatned by differentiation of volume. Esophageal pressure (Pes) was measured using
a saline-filled polye~hylene catheter (PE-90, 30 cm long), placed in the esophagus and
1 . 33
'-
-------------------------------------------
GP
A
B
c
TAB!IE 1. B100d Gases
JlCh (aq/a1)
Base 0.5 8 64
Base 4 32
Base 4 32
pH
7.4 7.35 7.20 7.20
7.30 7.33 7.30
7.36 7.34 7.40
pC02
43.2 42.7 43.6 39.2
39.5 35.2 36.8
42.2 36.7 35.8
p02
146 134 133 126
110 115 118
125 101
96
Reo -3
18.8 18.6 16.8 15.3
24.2 25.1 25.1
19.6 17.9 21.8
Blood gases are shown for 3 guinea pigs (A, Band C) used to assess the adequacy of mechanical ventilation during preliminary experiments. Values shawn are in ITUllHg.
Base = saline aerosol given.
34
rotameter
(J.>
CIl
,-""""'\
Ultrasonic Nebulizer (da Vilbl •• )
11ml/s
i-~-::""
Mechanical Ventilator Plethysmograph (Harvard)
BP
Ptr
+ • V
Ptp = P tr .• Pee
Fig. 3 Scheme oF Experimental Set-up.
connected to one port of a pressure transducer (Sanbom 267 AC). A liquid-filled
catheter is used here instead of an air-filled catheter because it is casier to fit a tube
without a balloon into the tiny esophagus of these small mammals. Without the baIloon
it becomes necessary to fill the tube with a liqUld to ensure proper transmission of the
pleural pressure. The tip of the esophageal catheter was placed in the lower esophagus
in such a way as to record a respiratory pressure with a cardiac artifact. Prc1iminary
experiments demonstrated that the tip of the esophageal catheter could be movcd a
centimeter or more without significantly changing the magnitude of the pressure
excursion. The other port of the pressure transducer was connected via a si de arm to the
tracheal catheter. The transpulmonary pressure (Ptp) was obtained from the differencc
between Ptr and Peso The functional residual capaclty (FRC) was maintained by
applying a positive end explfatory pressure (PEEP) of 3 to 4 cmH20 throughout the
experiment. The PEEP was applied by submerging the expiratory line from the
ventilator into 3-4 cm of water.
2.1.3 Methacholine Provocation Testing.
Aerosols were delivered by an ultrasonic nebulizer (deVilbiss) into the intakc port
of the ventilator. An airflow of Il mils was used with a nebuhzer output of O. J 8
ml/min. Aerosols were delivered for 30 s, starting with saline and continuing with
progressively doubling concentrations of Meh ranging fJOm 0.063 to 64 mg/ml. In
preliminary experiments animaIs mvariably showed a plateau on the conccntration
response curve with 64 mg/ml of MCh. For this reason this concentration of drug was
36
chosen as the maximal. The maximal resistance, or Rmax t is defined as the highest
response achicved before or after the last concentration of MCh delivered.
2.1.4 Experimental Protocol.
First measurements of pulmonary resistance were made during spontaneous breathing
to establIsh a stable baseline. Then each animal was paralyzed and mechanically
ventilated. Aerosols were administered and measurements were made again after saline,
and successive concentrations of MCh. Data were collected for 10 seconds and an
interval of approximately 3 minutes was allowed to elapse between aerosol applications.
After a complete concentration-response curve was established, the guinea pig was
exangl':!1ated and the lungs were removed and processed for histology and morphometric
studies.
2.1.5. Histologie and Morphometric Studies.
Immediately after the last concentration of MCh was delivered, the animaIs were
exanguinated and the lungs were removed, inflated and fixed with formalin (10%) at a
dlstending pressure of 25 cm H20 for 24-36 hours. Each Jung was eut into mid-sagittal
slices and cross-sections of the trachea, main bronchi, lobar bronchi and hilum were
obtaincd. Tissues were cmbedded 111 paraffin t eut into sections 5 um thick, and stained
with hematoxylin-phloxin-saffron (HPS) (108).
HPS stained sectIOns from both the left and right lungs were examined by light
microscopy (Zeiss, Laborlux S, W. Germany). Measurements were made by projecting
37
..
photographie slides of the airways onto a computer-controlIed digitizing board (Jandel
Scientific, model 2210-0, CA, USA). A digitizing program (Sigma Scan) was uscd for
all measurements. In 8 of the guinea pigs ail of the midsagittal alrways photographcd
were analyzed. ln the other 5 animaIs a sam pIe of 12 to 16 airways from mldsagittal
sections were analyzed. AH of the airways were photographed, but for any two or more
airways of similar size in a given field, only one was analyzed. Extraparenehymal
airways were measured in aIl of the guinea pigs. Airways whose maximum lo mInimum
internai diameter was equal to or larger than 2 were considered to be cut obhquely and
were not measured. The following parameters were measured in ail of the airways: the
internaI area (Ai) defined by the basement membrane (BM) , the external area (Ae)
defined by the outer border of the airway wall, the area of smooth muscle (SM) and the
length of the basement membrane (LBM). Airway wall area was then calculatcd, AW
= Ae - Al.
2.2 Quick-freezing of guinea pig lungs.
The second series of experiments was performed to examine the distribution of
bronehoconstrietion and to determine the importance of airway smooth muscle as a
determinant of heterogeneity.
2.2.1 Animal Preparation.
Six male guinea pigs (weight 350 - 400 g) were anaesthetized with urethane (50%,
l.5 g/kg i.p.) and instrumented as before. The animaIs were mechanieally vcntilated
38
(f=60 bpm, Vt=5 ml/kg). However the thoracic cavity was opened widely to facilitate
rapid freezing and removal of the lungs from the chest cavity.
2.2.2 Experimental Protocol.
Each animal was chaIJenged with MCh. Aerosols of MCh were delivered using the
method previously described (section 2.1.3) except that a more limited range of
concentrations of MCh was u~ed (32 to 128 mg/ml). These concentrations resulted in
a plateau (Rmax). Each animal was th en allowed to recover until it reached a pre-
determmed value of resistance along its rOl1centration-response curve. For 5 of the
guinea plgS the responses were chosen to be 100%, 75%, 60%,40%, or 15% of the
Rmax. The control animal received an aerosol of saline. Measurements of lung
resistance were made about every 2 to 5 minutes for the animaIs who se lungs were to be
frozen at high levels of bronchoconstriction (100, 75, 60 % Rmax) , and every 5 to 10
minutes for those fixed at low level of bronchoconstriction (40% and 15 %Rmax).
2.2.3 Histologie and morphometric studies.
Once the predetermined degree of bronchoconstriction was reached the animaIs were
exanguinated, the trachea was clamped at FRC maintained by a PEEP of 3 to 4 crnH20,
and the lungs were irnmersed in liquid nitrogen for 30 minutes. The lungs were
subsequently fixed in Carnoy's solution (70% ethanol, 20% chloroform, 10% acetic acid;
-80·C 18h, -20°C 6h, 4°C 4h) and transferred to absolute alcohol (4'C, 24 h). Paraffin
blocks were made from multiple sagittal slices of each lung. Sections 5 um thick were
39
eut and stained with hematoxylin and eosin (H&E).
Morphometric measurements were made as before, .lut this time the airways were
projected directly from a glass slide onto a computer-controlled digitizing board lIsing
a camera lucida apparatus (Zeiss Wetzlar, Germany). In ail airways the follow1I1g
parameters were measure<Ï: the internaI or luminal area (Ai) defined by the epithcltum
(EP), the external area (Ae) defined by the outer border of the airway wall, the length
of the base ment membrane (LBM), the length of the epithelium (LEP), and the arca of
smooth muscle (SM) where appropriate. Since the objective of this section was to
measure airway narrowing, the length of the epithelium rather than the basement
membrane was used in most cases to assess the size of the lummal arca, even though
LEP was only 3 to 5 % less than LBM. Also, the correlation between these two
parameters was good (r=0.99); at times LEP and LBM may be uscd intcrchangcably.
The variability between this method and that of projecting photographlc shdes onto the
digitizing board (see section 2.1.5) was less than 4 %. The correlation coefficIents were
0.99, 0.96, and 0.97, for measurements of basement membrane, smooth mm~c1e, and
airway wall respectlvely.
2.3 Frequency-response characteristics of the volume and esophageal pressure
measurement systems.
The frequeney response of the Plexiglas box was tested using a loudspcaker and sine-
wave generator to ereate a sinusoidal pressure signal. A pressure transducer (Validyne
MP-45 + 5 cmH20) was used to measure input pressure to the plethysmograph and a
40
similar pressure transducer was used to measure the output pressure. The pressure
sIgnais did nol show a significant difference in amplitude or phase up to a frequency of
5 Hz.
To verify that air did not leak out of the box, a known volume of air was injected
into the box through an entry port and compared with the change in volume recorded
from the pressure transducer. The time constant was obtained by following the time
course of the decrease in the volume signal and was > Imin. This check was performed
at the beginning of each experiment.
The time constant of the catheter-transducer system used to measure Pes was
determined by examinmg the time course of the pressure signal after the application of
a step change in pressure. The tracheal catheter was placed in a 60 ml container whose
Iid was a latex membrane that could be inflated to a constant pressure using a compressed
air source. When the balloon was punctured the time course of the explosive decrease
in the pressure signal provided a time constant of 26 ms for Peso
2.4 Data Collection.
Volume and pressure signaIs sensed by the transducers were fed into an eight-channel
strip-chart recorder (Hewlett-Packard, model 7758B) where the signaIs were amplified
by electronic carrier pre-amplifiers. These were used due to their high gain with high
amplifier stabihty (Physiologie Recording by D.L. Fry, Physiol.Rev. 40:753-88,1960).
Electrical signais were then sampled at 60 Hz with a 12-bit analog-dlgital converter (DT
2801-A, Data Translation, Malborough, MA) and stored in a computer (Compaq
41
"
DeskPro 286) for further analysis (See Data Analysis).
2.5 Data Analysis.
2.5.1 Physiological measurements.
The mechanics of the respiratory system were evaJuated usmg the following cqllatlon:
. Ptp=EV+R,V+K
where Plp = transpulmonary pressure E = elastance V = volume ~1. = Jung reslstance V = tlow rate K = constant
This equation describes a linear, single-compartment modeJ of the lung. The paramclers
are obtained by multiple linear regression where the least squares method 15 lI~cd to
determine the best fit of the equation to the data. The Jung is assul11cd 10 bchave
linearly, so that E and Rare mdependent of volume and flow. The prc~sllre los ... cs duc
to inertia ue assumed to be negligibly small. Il is also assumed that E and R arc
independent of frequency (87). The constant k allows for the case wherc lCro volulIIC
is not the point at whlch there is zero elaslic recoil prc~~ure lI1slde the lungs (thls Illay
happen if the expiratory lIme IS nol long enough, givmg nsc to mtrinslc PEEl> (68».
Ca\cuJatlOns of RI were carried out after data sels were cn~emble avcraged
employing an algorithm that used peak expiratory flow to identlfy mdlvldual breaths.
E and R were caIculated from each breath that was averaged. The re~ulling valL1c~
42
represented the average of 5 to 10 breaths + SEM. The resistance of the esophageal
catheter was 0.14 cmH20/ml/s and was subtracted from RL•
This analysis was done using the software Anadat, written by J.H.T. Bates, and an
algorithm written by D.H. Eidelman, at the Meakins-Christie Laboratories, McGiIl
University.
2.5.2 Morphometry.
AJI morphometric measurements were made by a single observer. However, the
technique was validated by measuring both intra- and inter-observer variability. For
verifying intra-observer variability, the same person performed measurements of the
same auways on two different occasions. Identity plots of smooth muscle, base.:lent
membrane and airway wall yielded r values of 0.99, 0.98, and 0.99 respectiveJy. Both
intra- and inter-observer variabiJity wert" Jess than 2% for ail parameters measured. In
formalin-fixed lungs, é1 total of 429 airways were measured, including intra- and extra-
parenchymal, with an average of 33 airways per animal (range 19 to 46). In liquid
nttrogen-tixed lungs only mtraparenchymal airways were measured with an average of
77 airways measured per animal (range 64 to 106) for a total of 462 airways.
In formalin-fixed lungs intraparenchymaI airways were divided into three groups
based on the Jength of the basement membrane: small (lbm= 0-0.99 mm), medium
(lbm= 1.0-1.99 mm) and large (lbm= 2.0-2.99). Intraparenchymal airways whose Ibm
was greater than 3 mm were rare, and not present in ail of the lung sections.
Intraparenchymal airways were also divided accordmg to the median airway in each ., .
43
-.
animal in order to compare similarly sized airways. These airways were classi fied
according to the presence or absence of cartilage from visual inspection of the
histological sections. In frozen lungs the intraparenchymal airways wcrc divlded into
small, medium, and large, and into small and large based on the median airway. The
smooth muscle was standardized by dlviding by the ideallummal arca (fLA) to adjust for
differences in airway size. ILA was calculated from Ibm, assuming that the
unconstricted airway was a perfeet circle.
The percent of smooth muscle (%SM) shortening was calculated by obtainmg the
circumference of the mean area of airway smooth muscle in 1) the relaxed, or Ideal,
state, and 2) the contracted, or the observed, state. The circumference was calculatcd
from the measured smooth muscle area simply by mu)tlp)ying the area by 2 and dlviding
it by the radius of the area. The Ideal length was obtained by calculating thc area
enclosed by the smooth muscle assuming the unconstncted alTway was a perfeel clrele.
This was done by adding the ideal luminal area (section 2.5.2) to the area cl1closed
between the epithelium and the outer border of smooth muscle. From thls area, the idcal
length was derived. Similarly, the actuallength of ASM was obtained from the observed
area enclosed by ASM in the constricted airway. The %SM shortenmg was then glVCI1
by:
ideal length of ASM - actual length of ASM %SM - x 100
ideal length of ASM
44
2.S.3 Statistical Analysis.
Comparisons of means among groups of different sized airways for the 13 guinea
pigs were made using analysis of variance, and tests for multiple comparisons. P values
< 0.05 werc considered significant. Data are expressed throughout as mean + SEM.
(Standard Error of the Mean). SEM are shown throughout unless otherwise stated.
Correlations between smooth muscle and either resistance or sensitivity were done by
Iinear regression using the least squares technique.
ln order to examine the distribution of bronchoconstriction, cumulative frequency
distribution curves were built for the total of the airways, and for small and medium-
sized airways. The number of large airways was too small for a meaningful analysis.
Comparisons of these cumulative distributions was done by the Kolmogorov-Smimov
two-sample test.
.f
.1
45
1 Chapter 3
RESULTS
1. Methacholine (MCh) Provocation Test.
3.1 Physiological measurements.
Table 2 shows the results of lung resistance (RI) in cmH20/ml/s obtained for 13
guinea pigs challenged with MCh. Resistances are shown first for basehnc breathing,
then for values recorded as each animal was administered progressively doubling
concentrations of MCh. The 5tandard errors of the mean were negligibly small so they
have been omitted from the table. The mean baseline resistance was 0.18+0.09
cmH20/ml/s (+SEM, n= 13) during mechanical ventilation, wlth valucs rangll1g
somewhat among animaIs from 0.08 in guinea pig #1 to 0.35 ln guinea pig #2, a 4.4 fold
difference. There was httle or no variabihty In the baseline withm thc samc animal.
Figure 4 shows a representative concentration-response curve for gUInca pig # 10.
Values of lung resistance have been plotted against the MCh concentration. The basclll1c
resistance was 0.14 cmH20/ml/s. After the administration of MCh aerosoIs, the responsc
increased gradually until a maximal response was reached, followmg the expccted
sigmoid shape of the c1assical concentration-response curve. Tht., threshold response
occurred al an approximate concentration of 2 mg/ml of Meh. Thc maximal rcsi:.- ~"CC
reached on the plateau of the concentration response curve, or Rmax, was 2.27
cmH20/mIls, a 16.2 fold increase from the baseline. The effective concentratJOn
46
"""" -..J
~ .. w~ ;~
TABLE 2. Lung re.i.tance in guine. pigs 1 to 13 exposed to .erosols of .ethacholine for co.plete coneentrationrespon.e eurv.s.
AEROSOL GP1 GP2 GP3 GP4 GP5 GP6 GP7 GP8 GP9 GP1- GP11 GP12 GP13 (mg/ml)
,,-
BASE 0.08 0.35 0.19 0.34 0.17 0.14 0.29 0.22 0.12 0.14 0.10 0.11 0.12
0.06 0.12 0.43 0.19 1.14 0.17 0.16 0.30 0.29 0.12 0.17 0.10 0.12 0.13
0.13 0.15 0.49 0.20 3.82 0.18 0.16 0.31 0.29 0.38 0.19 0.10 0.13 0.14
0.25 0.31 0.50 0.23 2.61 0.19 0.19 0.31 0.32 0.19 0.19 0.11 0.14 0.14
0.5 0.06 0.54 0.28 2.31 0.23 0.23 0.31 0.33 0.23 0.24 0.20 0.22 0.16
1 0.71 2.04 0.61 2.05 0.28 0.27 0.41 0.35 0.25 0.23 0.36 0.61 0.20
2 1. 61 2.41 1.41 2.18 1.26 2.41 0.47 0.31 0.29 0.25 0.70 0.56 0.25
4 1.38 2.15 2.17 2.15 2.46 3.57 2.54 0.30 0.31 0.64 1.03 1.04 0.57 1
8 1.55 2.65 2.41 2.14 3.81 3.14 3.13 0.44 0.39 ] .3:;: ?.06 1.07 0.61
16 1.14 3.03 2.29 2.36 4.50 2.77 3.65 1.15 0.66 ) r· .... ,'.Jo. 2.96 1.01 0.77
32 0.86 3.42 2.37 2.57 4.52 2.66 3.87 2.16 C.90 2.26 3.20 1.21 0.98
64 1.08 3.60 2.45 ~.139 4.09 2.62 3.96 7.34 1.5~ ') .2 ! 4.01 1..33 1.24
---The values of lung resi9tance are tn cmHp/ml/s and repreeent the average (If 5 CI) 10 breaths. SEM wer€ negligible, ~Ses A~ction 2.5.1}. GP = gu~nea pig.
Fig. 4 Representative concentration-response curve, showing lung resÎstance {RJ versus the methac.holine concentration for guinea pig #10. (B=baseline). Each dot represents the average resistance of 5 to 10 breaths. SEM were smaller than the size of the symbols.
r
Guinea Pig # 10 4
~
fil Rmax = 2.27 cm~O/ml/8 "'-~ a 3 ECao = 6.68mg/ml
"'-0 N .-.
== / a 2 • (,) 1 "-"
e ~ 1 / ~
• e_-e----e--e
/ 1--...1
B 0.06 0.25 1 4 16 64
Methacholine (mg/ml)
r
48
required to reach haIf of the maximal response, the ECso or sensitlvity, was 6.68 mg/ml.
The values of Rmax and ECso for the 13 guinea pigs are shown in table 3. Ali of
the animaIs reached a maximal responsc on their concentration-response curvcs. The
Rmax varied among animaIs, ranging from 1.24 in guinca pig #13 to 4.52 cmH10/ml/s
in gumea pig #5, a 3.6 fold difference in the range of responsivcncss among '1nllnals.
The ECso showed greater variability from 0.09 in guinea pig 114 to 22.63 mg/ml in guinca
pig #9, a 254 fold difference between the least and the most sensitive anllnal. The
coefficient of vanation for Rmax was 0.41. There was no relationship betwcln the body
weight and either the baseline resistance (r=0.341), or Rmax (r=0.08). Also there was
no significant correlation betweer. Rmax and the baseline resistance (r=0.427), or
between Rmax and the ECso (r=0.069).
3.2 Morphometry.
Figure SA is a photomicrograph (x25) of an intraparenchymal airway from guinea
pig #2, whose length of basement membrane, Ibm, is 1.03 mm (or 0.33 mm in
diameter). Hs smooth muscle standardized for size 0.09. Note that the alrway smooth
muscle (ASM) when standardized for size is divided by mm2 and therefore has no umts.
The corresponding image of the traced outlines of the structures of interest are shown in
tigure SB. The epithelium is the outermost layer adjacent to the lumen. Next, is the
basement membrane on which the epithelial cells he. A thlck layer of smooth muscle
surrounds the epithelium, and it appears to be continuous; this was true in most of the
airways sampled. The airway wall is defined as the structure bounded the basement
49
.
reqUlre<! to reach half of the maximal response, the ECso or sensitivity, was 6.68 mg/ml.
The values of Rmax and ECso for the 13 guinea pigs are shown in table 3. AlI of
the animais rcached a maximal response on their concentration-response curves. The
Rmax varied among animais, ranging from 1.24 in guinea pig #13 to 4.52 cmH20/ml/s
in gumea plg #5, a 3.6 fold difference ln the range of responsiveness among animaIs.
The ECso showed greater variability from 0.09 in guinea pig #4 to 22.63 mg/ml in guinea
plg #9, a 254 fold difference between the least and the most sensitive animal. The
coefficient of variation for Rmax was 0.41. There was no relationship between the body
welght and either the baseline reslstance (r=0.341), or Rmax (r=0.08). Aiso there was
no slgnificant correlation between Rmax and the baseline resistance (r=0.427), or
between Rmax and the ECso (r=0.069).
3.2 Morphometry.
Figure 5A is a photomicrograph (x25) of an intraparenchymal airway from guinea
pig #2, whose length of basement membrane, Ibm, is 1.03 mm (or 0.33 mm in
diameter). Hs smooth muscle standardized for size 0.09. Note that the airway smooth
muscle (ASM) when standardized for size is divlded by mm2 and therefore has no units.
The corresponding image of the traced outlines of the structur"!s of interest are shown in
tigure 5B. The epithelium is the outermost layer adjacent to the lumen. Next, is the
basemelll membrane on whlch the epithelial cells lie. A thick layer of smooth muscle
sllrrollnds the epithelillm, and it appears to be continuous; this was true in most of the
airways sampled. The alrway wall is defined as the structure bounded the basement
49
1 TABLE 3. The weiqht, maximal resistance and sensitivity for guinea pigs (n=13) challenqed with methacholine.
GUINEA PIG WEIGHT Rmax ECSO loqECSO
NUMBER (g) (mg/ml)
1 364 1.61 0.96 -0.05
2 407 3.60 0.90 -0.16
3 375 2.45 1. 71 0.78
4 356 2.89 0.09 -3.55
5 328 4.52 3.56 1 .83
6 326 3.57 1. 64 0.'/1
7 306 3.96 3.32 1.73
8 318 2.34 16.22 4.02
9 420 1. 56 22.63 4 .50
10 374 2.27 6.68 2.7'1
11 420 4.01 7.73 2.95
12 290 1.33 2.35 1.23
13 324 1.24 8.34 3.06
Rmax = maxima l res istance in cmH~O /ml / s. (This va lue represents the average of 5 te 10 breaths. See section 2.5.1).
EC5U = the ef fecti ve concentration at which 50% of the maxima l response is achieved, in mg/ml.
... logEC5o = logarithm of the EC50 •
50
J
Fig. SA Pholomicrograph of guinea pig airv .. ay (x25) approximately 0.33 mm in diameter
51
• .,If , l'
, 1..,/ , ' ..
. ... .....
,
Fig. SB l'racine of guinea pig airway shown in fig. 5A. The stru\:tures labelled are the airway wall (A W), the smooth muscle (SM), the basement membrane (BM), and the epithelium (EP).
52
l
ft
Figs. 6A and 6B Photomicrographs of airways from guinea pigs 1 and 3 respectivel y ShOWÎllg differences in ai.rway smooth muscle among different animals.
53
1
Figs. 7A and 78 Photomicrographs of airways from guinea pig 5 showmg differences in the amount of alrway smooth muscle within the same animal. (Also see fig. 7C).
54
, -.
, , . . ~ .. • 1 "
• ,); 1 \~ • , .. ~\,4 ~I ~'
" .... '.l , ''',.
~'.'
, ... . ( -Il,
, . '. ..... \ \ .
" '~\' . ' .. '" "." .. ~ . ,. ,,' 'l \ 'II. • ..
\~4. \'. • . , lA 'It .... • .. ,.. .. .j ., 't
.1" • ... , .... .
Fig. 7C Photomicrograph of another airway from guinea pig 5 showing a larger amount of airway smooth muscle than in figs. 7 A and 7B.
ss
_l"l;>
membrane of the epithelium and the border with the parenchyma.
Figures 6A and 6B are photomicrographs (x25, xlO) from guinea plgs 1 and 3
respectively, which show dlfferences in the amount of smooth muscle bctween different
animais. Smce sorne of the alrways are constneted, the visual cffeet is to sec more
muscle. But that is irrelevant since the smooth muscle measurement is eorrcctcd for
airway size and is divided by the Ideal luminal area. Figure 6A is an example of another
airway, 0.58 mm in Ibm, and 0.22 of smooth muscle showing about 2.4x more smooth
muscle than that 111 figure 5. The airway ln figure 6B is 0.66 mm III Ibm, and has 1.18
units of smooth muscle whlch IS about 5.4x larger than that in ligure 6A. and can he
secn as a thick layer surrounding the airway lumen. Note that in figure 613 the airway
epIthehum has become infolded, reducing the area of the lumen.
Differences in the amount of smooth muscle are also evident wlthlll the same animaI.
Figures 7A, 78 and 7C are photomlcrographs (x25, x25, xW) of alrways from guinca
pig 5. In the fifst two, the alrways have about the same Slze, namely 0.71 and 0.76 mm
in Ibm; lhey also have about the same amount of smooth muscle, 0.24 and 0.21
respectlvely. But the airway in figure 7C has about tWlce the amount of smooth muscle
wJth 0.42; thls alfway IS also an example of an mtraparenchymal cartJlagll10us alrway,
of 1.23 mm in Ibm.
3.3 Distribution of airway size for airways sampled.
Figures 8 and 9 show the distribution of airway sire for the airway~ samplcd. The
estimate of slze is based on the length of the basement membrane for cach of the 13
56
Fig. 8 The distribution of intraparenchymal airways sampled (n=36) for each of 13 guinea pigs according to the length of the bé!scment membrane (mm). The number of airways per animal is indicated in brackets. There was a wide range in the size of the airways sampled in each animal, from 0.25 in guinea pig 4 to 2.94 mm in guinea pig 13.
4
-s a -G (37) (15) (~5) (23)
(~ 1) s= 3 .. • • • ... (33) (35) • oC • a • • • • (33) (22) • G • • :II • (l·u (28) • • • (22) • ~ • et • • • • • • = 2 • • • ! G • • • • • • • • s • (.1) • • • • • • 1 G • 1 • • •
1 • 1 ID • • 1 1 • .. • • 1 • = • • • 1 • • • • 1 • • • • 1 1 • ct
1 .... • • • • 0 • 1 • 1 • • • • 1 1 1 • 1 ~
1 1 1 1 • 1 • • ; .. 1 • = 1 • 1 1 • • • • G • • • • 1 • • ~ • 1 •
0 -l_ -L-----L -l_-J. __ l __ -'
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Guinee. }lig Number
57
,
Fig. 9 Distribution of the number of airways per size cJas'i according tu the length of the base ment membrane (Ibm). Intraparenchymal (l) airways (n=361) weie divided into small (lbm=O-O.99 mm), medium (lbm= 1-1 99 mm) and large (lbm=2-2.99 mm). Extraparenchymal (E) airways (n =68) Y/ete 2-6 mm in Ibm. The number in brackets is the number of animaIs ttiat hlid the same number of airways for a given size interval .
•
..
40
• 30
~ • i .... • 0 20 • ,
l ~ • (1) \
j • ~ >
1 l
" ., Z 1
!
1 (1) 1 (3) 10 • • (1)
(1) • • (1) 1 (1) (1)1 10) (01 (0) (1) (a)
t· (1) ~ 0 .,
0-0.99 1-1.99 2-2.99 2-8 (1) (1) (1) (1)
Lenath of buement membrane (IIUD)
t i t r ft l>
1 , ~
& '" ., ~ ~ ,
58
gumea plgs. A total of 429 airways were measured, 361 were intraparenchymal and 68
were extraparenchymal. The number of airways ranged from 14 in guinea pig #8 to 41
in guinea pig #4. There was a wide range in the size of the airways sampled within each
animal, From 0.25 mm in guinea pig #4 to
2.94 mm JO guinca pig #13, as measured by the length of the basement membrane or
approxlmatcly 0.08 to 0.94 mm ;0 diameter (fig. 8). There was a significant difference
in airway slze of Jntraparenchymal airways among animais (p< 0.01, ANOVA), but there
was no difference JO the extraparenchymal airways. This does not mean that sampling
has failed, smce different animais can exhibit differences in the geometrical dimensions
of the airway tree.
As expected, the maJority of airways analyzed were small; 76% of the airways
(n=328) were ln the range of Ibm <2 mm. This point is further illustrated in figure 9.
The largest number of airways had a basement membrane Iength less than 1 mm
(n=192), followed by the airways whose Ibm was less than 2 mm (n=136). Few
airways were found between 2 and 3 mm (n=33). About 16% of the airways (n=68)
werc extraparenchymal. In the mtraparenchymal airways the mean Ibm was 1.11 +0.05
mm, whereas in the extraparenchymal airways it was 4.93+0.17 mm (±SEM) (table 4).
Because of the discrepancles in the distribution of airway size among animaIs, the
alrways were divided into vanous size categories in order to make sui table comparisons
of alrway smooth muscle.
59
'--
TABLE 4. The Mean size of airways in formalin-fixed lungs based on the length of the basement membrane (LBM).
GP # IHTRA n EXTRA n TOTAL n
1 1.05 + 0.09 33 4.55 ± 0.90 3 1.35 + 0.20 36
2 1.07 ± 0.10 35 6.07 ± 1.00 3 1.47 ± 0.25 38
3 1.01 ± 0.12 37 6.18 ± 1. 23 4 1.55 ± 0.29 41
4 0.70 ± 0.05 41 5.01 ± 0.49 5 1.16 ± 0.21 46
5 0.98 ± 0.08 33 5.33 ± 0.59 5 1.55 ± 0.26 38
6 1. 03 ± 0.13 22 5.16 + 0.49 4 1.69 + 0.33 26 <J' <::> 7 1.39 ± 0.16 15 4.46 ± 0.33 9 2.54 ± 0.34 24
8 1.06 + 0.15 14 4.74 + 0.32 5 2.03 ± 0.23 19
9 1.27 ± 0.15 25 4.84 ± 0.63 6 1.94 ± 0.30 31
10 1.27 + 0.12 23 4.43 ± 0.42 6 1.93 ± 0.27 29
11 1.37 ± 0.08 26 4.74 ± 0.49 7 2.09 ± 0.27 33
12 1.12 + 0.09 22 4.59 + 0.60 6 1.84 ± 0.30 28
13 1.13 ± 0.10 35 4.05 ± 0.34 5 1.89 ± 0.18 40
TOTAL 1.11 ± 0.05 361 4.93 ± 0.17 68 1.77 ± 0.10 429
Values of LBM are expressed as means in mm ± SEM. n = number of airways. INTRA=Intraparenchymal airways. EXTRA=Extraparenchymal airways.
3.4 Measurement of airway smooth muscle.
3.4.1 Airway !lmooth muscle as a function of airway size.
Mca~urcmcnt~ of alr'Nay smooth muscle are shown in tables 5 through 9. The results
are reportcd for intrapare'lchymal (1) and extraparenchymal (E) airways (table 5). The
amount of ~mooth muscle In intraparenchymal alrways (0.18 +0.01) WaS significantly
(p <0.01 two-way ANOVA) hlgher than that in extraparenchymal airways (0.05 +0.01).
The mtraparenchymal airways were subdivlded lOto two groups according to the
mcdlan alrway (1., =alrways less th an the median; IL =auways greater than the median)
for each ammal in order to compare similarly sized airways (table 6). In Is the amount
of smooth muscle (0.19 +0.01) was not slgnificantly different (p>0.05) than that in IL
(0.17+0.01). The smooth muscle in both Is and l, was significantly differei,t (p<O.Ol
ANOVA) th an that III the extraparenchymal alrways.
Intraparcnchymal alrway~ were also dlvlde.d JOto small (Ibm =0-0.99 mm), medium
(Ibm= 1-1.99 mm), and large (lbm=2-2.99 mm) for convenience (table 7). The amount
of slliùoth muscle was the same ln small .'Id medium sized airways with 0.18+0.01.
This éimount was hlgher than that of large airways (0.14+0.02), but the difference was
Ilot sigllltïcant (p>0.05).
furtlu:'-morc, the intraparenchymal alrways were divided into membranous or
carulaglllous based on the presence of cartilage (table 8). The amoun! of smooth muscle
was sllghtly less III membranous alrways than in cartilaginous airways (0.17+0.01 versus
O.22±0.02) but the difference was not slgnificant (p:> 0.05).
FlIlally, the amount of sl1100th muscle was tabulated by extracting the top 25 % of the
61
~.
TABLE S. Amount of airway smooth muscle (ASM) in intraparenchymal, extraparenchymal and in the total of the airways.
GP # INTRA n EXTRA n TOTAL n
1 0.14 + 0.02 33 0.04 ± 0.01 3 0.13 ± 0.02 36
2 0.15 + r.02 35 0.03 + 0.01 3 0.14 ± 0.02 38
3 0.27 + 0.04 37 O.OJ + 0.01 4 0.26 ± 0.04 41
4 0.20 .± 0.02 41 0.03 ± 0.01 5 0.18 ± 0.02 4G
5 0.19 + 0.03 33 0.03 ± 0.00 5 0.17 ± 0.03 38
6 0.20 ± 0.02 22 0.03 .J... 0.01 4 0.18 ± 0.03 26 ~ N
7 0.17 + 0.02 15 0.06 ± 0.01 9 0.13 + 0.02 24
8 0.22 ± 0.02 14 0.05 ± 0.01 5 0.17 ± 0.03 19
9 0.17 ± 0.02 25 0.07 ± 0.02 6 0.16 ± 0.02 31
10 0.14 ± 0.01 23 0.06 ± 0.02 6 0.12 + 0.01 29
11 0.17 + 0.01 26 0.06 ± 0.01 7 0.14 ± 0.01 33
12 0.23 2:. 0.02 22 0.08 + 0.02 6 0.20 + 0.02 28
13 0.10 .± 0.01 35 0.04 .± 0.01 5 0.09 + 0.01 40
TOTAL 0.18 + 0.01 361 0.05 ± 0.01 68 0.16 ± 0.01 429
Values of ASM are expressed as means .± SEM and have no unlts. n = nurnber of alrways. INTRA=Intraparenchymal airways. EXTRA~Extraparenchymal airways.
0\ w
TABLE 6. Airway smooth muscle (ASH) in intraparenchymal airways divided according to the median airway of each quinea pige
GP # I8 n IL
1 0.13 + 0.02 16 0.15 + 0.03
2 0.18 ± 0.04 17 0.13 ± 0.02
3 0.32 + 0.07 18 0.21 + 0.04
4 0.21 ± 0.03 20 0.18 ± 0.03
5 0.21 + 0.05 16 0.17 + O.OJ
6 0.19 ± 0.03 11 o . 23 -'- 0.05
7 0.18 + 0.05 7 0.17 + 0.02
8 0.20 + 0.03 7 0.24 + 0.04
9 0.17 + 0.03 12 O.lb ~ 0.02
10 0.1] + 0.01 Il 0.14 T 0.02
Il 0.1'1 + 0.02 13 0.17 ± 0.02
12 0.25 + 0.02 11 0.21 + 0.02
13 0.11 + 0.01 17 0.09 ± 0.01
TOTAL 0.19 + 0.002 176 0.17 ± 0.01
Values of ASM are expressed as means ± SEM and have no units 18 = intraparenchyrnal airways less than the rnedian airway IL = intraparenchymal airways greater than the median ajrway
n
17
18
19
21
17
11
8
7
1.3
12
13
11
18
185
~~ ......
TABLE 7. Amount of airway smooth muscle CASH) in small, medium, and large intraparenchymal airways.
GP # SMALL n MEDIUM n LARGE n
1 0.15 + 0.03 20 0.15 ± 0.03 la 0.08 ± 0.01 3
2 0.18 + 0.04 17 0.13 ± 0.03 15 0.11 ± 0.01 3
3 0.31 ± 0.05 78 0.15 + 0.02 5 0.08 + 0.01 4
4 0.19 ± 0.03 35 0.26 ± 0.04 6 0
5 0.21 + 0.04 22 0.15 ± 0.03 9 0.13 ± 0.01 2
6 0.19 + 0.03 12 0.27 + 0.05 8 0.08 + 0.02 2 <l' ~
7 0.19 + 0.06 6 0.17 + 0.02 8 0.12 1
8 0.18 ± 0.03 8 0.26 + 0.03 5 0.31 1
9 0.18 ± 0.03 10 0.16 + 0.02 10 0.14 + 0.02 5
la 0.13 ± 0.01 10 0.14 ± 0.02 10 0.15 ± 0.02 3
Il 0.14 ± 0.02 6 0.17 + 0.02 18 0.24 + 0.02 2
12 0.23 ± 0.02 8 0.23 ± 0.02 11 a
13 0.10 -+- 0.01 la 0.11 + 0.01 18 0.08 ~ 0.02 7
TOTAL 0.18 ± 0.01 192 0.18 ± 0.01 136 0.14±0.02 33
Values of ASM are expressed as rneans = SEM and have no units. n = nurnber of airways. Small: Ibm=0-0.99mmi nedlum: Ibm=J --1. 99mmi large:lbm=2-2.99mm.
____ <~ .............. .u ........ ------------------~----------------------------------------------
TABLE 8. The amount of airway smooth muscle (ASM) in intraparenchymal airways divided accordinq to the presence of cartilaqe.
GP # Membranous n cartilaqinous n
1 0.13 .± 0.02 26 0.20 + 0.06 7
2 0.14 + 0.02 27 0.39 ± 0.07 B
3 0.28 + 0.05 27 0.25 =- 0.03 10
4 0.17 ± 0.0:' 31 0.28 ± 0.06 10
5 0.19 ± 0.03 27 0.14 ± 0.05 6
6 0.19 ± 0.02 17 0.26 ± 0.08 5 a.. Ul
7 0.14 + 0.01 10 0.19 + 0.n3 5
8 0.21 ± 0.02 9 C.23 ± 0.0'5 5
9 0.17 ± 0.02 17 C .15 + 0, l:;J 8
10 0.]4 ± 0.01 18 0.14 ± 0.02 5
11 0.14 ± 0.01 12 0.19 ± 0.[;2 14
12 0.20 + 0.02 15 0.29 :t 0.03 7
13 0.10 ± û.lll 21 O.] 1 J: 0.02 14
TOTAL 0.17 ± 0.01 257 C.22 ± O.O? 104
Values of ASM are expreesed as means in ±SEM (no units) . n = nurober of airways
.' ""'1
1
1 ~-_ ..
--------------------------------------~'~~------------
TABLE 9. ASM IN THE TOP 25% OF THE INTRAPARENCHYMAL AIRWAYS
Guinea pig # ASM (X ± SEM) n
1 0.12 + 0.03 8
2 0.13 + 0.03 9
3 0.12 + 0.02 9
4 0.25 + 0.04 10
5 0.1:' + 0.04 8
6 0.22 + 0.07 6
7 0.18 + 0.04 4
8 0.25 + 0.03 4
9 0.13 + 0.02 6
10 0.12 + 0.02 6
11 0.18 + 0.04 -;
12 0.22 + 0.03 6
13 0.09 + 0.01 10
TOTAL 0.17 ± 0.02 93
The amount of airway smooth muscle (ASM) is expressed as the means ± SEM and has no units.
n = nurnber of airways
66
mtraparenchymal alrway~ m an altempt to see whether the larger alrways correlated
beltcr wlth the mdlce~ of responslveness, since these airways contribute most to Jung
resistancc (table 9). The smooth muscle here was 0.17 +0.02, which is comparable to
that found in ail of the mtraparenchymal airways.
3.4.2 Variability in airway smooth muscle among guinea pigs.
Figure 10 shows the distribution of airway smooth muscle for intra- and
extraparenchymal
alrways among the mdividual guinea pigs (n = 13). There was a significant difference in
the amount of s:nooth muscle in intraparenchymal airways among guinea pigs (p<O.Ol,
ANOV A) regardless of how the alrways were dlvided. This finding was not observed
111 cxtraparellchymal alrways. The mteranimal variability was similar in both groups.
The :imooth Illuscle vaned l'rom 0.10+0.01 in guinea pig h'13 to 0.27+0.04 in guinea
pig #3 III IIltraparcnchymal alrway!!, and it varied from 0.03+0.01 in guinea pigs
2,3,4,5, and 6 to 0.08+0.02 In guinea pig #12. In both groups the range represents a
2.7 fold vanatlon ln alrway smooth muscle.
3.4.3 Variability in airway smooth muscle among different sized airways.
The dlstnbution of alrway smooth muscle in 3.11 of the airways sampled (n =429) has
bccn ploUet! agalllst alrway size determmed as the ideal luminal area (lLA) in figure Il.
This graph I~ a reprcscntatlon of the non-standardlzed smooth muscle so that airway size
has not been taken into account. Not surprisingly, the smooth muscle increases from the
67
/'
Fig. 10 The amount of airway smooth muscle (standardized lor dlrway S17e) iï. plottcd versus the guinea pig number, for Jn[ra- and cxt:ap~('llchyma1 ,Jrw'-"ys. A significantly (p < 0.05) lruger amount of smooth muscle "-as f,'"nd In
intraparenchymal airways of all animais. The mteran:mal vanabliily ""as si mitaI' in both groups of airways. Values are expressed as 3"!can .±. ~EM.
Il 0.31 ~ Co)
• :i :3 0.2 o o El fil
~ t 0.1 ~
0.0
~ 1 , 1
i
1
r I
1 1
_ :: intrllpare!lch.yœ.a1
o = ~%trl\pU'.nah)m al
.,. l -
1"
1" IJ r-
~
1 1"
l l ~ 1 1 2 3 4 5 6 7 8 9 10 11 12 13
Guinea Pig Number
68
Fig. 11 The distribution of airway smooth muscle (ASM) as a function of auv.'ay Sile.
as given by the idealluminal area (lLA) for a total of 429 airways In formalinfixed lungs (n= 13). The smooth muscle plotted here has not becn standardlzed for airway size, and as expected, it lOcreases from the smallest to the largest airways. 0 = Intraparenchymal él1rways. 0 = extraparenchymal ail'\\/ays.
~ 'Cf)
0.3
-< 0.1
0.0 0.001 0.01
------------------------- ------------------------
co
• • coo~ • • 0
co co co co
• _. 0 0 0 _ .. CD
_.- CI ct
•• • ':"" ce. - .:-. -- CL.cto·
~... • '10 " ... #e ...
co
0.1 10
69
smallest to the largest airways. Of note IS the substantial variabllity ln the quantlty of
ASM among airways of equivalent size.
The amount of smooth muscle Just described may be called the observed sl1100th
muscle. Assuming a linear relationshlp, ASM can be plottcd agalllst the Ideal IU1l1111al
area (ILA) to obtain the slope and intercept from which the prcdicted arnollnt of S1l1ooth
muscle may be derived. The predlcted value of smooth musclc IS thcn glvcn by
SM=O.113(lLA)+0.OO5 for intnl.~arenchymal airways, and SM=-O.OOl(ILA)+O.077,
for extraparenchymal alrways.
The absolute variabllity in airway smooth muscle IS given by the dlffercnce betwccn
the observed and the predlcted smooth muscle as shown in ligure 12A, whcre It 15 plot~ed
agamst the Ideal 1 11111 nal arca (lLA). Smce the value~ of smooth muscle have Ilot bcen
standardlzed for alrway Slze, the larger alrways show greatcr vanabIllty Figure 1213
shows the standardlzed, or relative, smooth muscle vanabJlity obtained by dlvld1l1g the
observed by the predlcted amount of ~mooth muscle. Here, it has bccn plouet! agamst
the ideal lumll1al area, showmg that the smooth IllUSCIe has a hetcrogcneolls dlstnbutlon
across airways of dlfferent Slzes, whelher Il be II1tra- or cxtraparenchymal alrways.
3.5 Correlations betwcen smooth muscle and Rmax and logECso '
3.5.1 Intraparenchymal airways.
There was no correlation betwcen the smooth muscle of 1I1traparcnchymal illfways
and the Rmax (r=0.117) (ligure 13A). Likewlse, no correlation was round bctwccn
smooth muscle in any of the small (r=0.136) or medium (r=O.072) Sllcd alrways and
70
Fig. 12A The smooth muscle (SM) absolute (Abs) variabllity obtained from the difference of the observed and predicted amount of smooth muscle is plotted against the ideal luminal area. fi is the total number of airways for the 13 guinea pigs challenged with methacholine to obtain complete concentratlOnresponse curves. The distribution of this variability is shown for intra- and extraparenchymal airways.
"
."
Intraparenchymal n = S61
CIJ~ 0.1
a a ~ ..... .-4 9 ~ 0.0 ~
~ CIl
~ ::; CI.J
-0.1 0.001
• • •
J- ••••
• .' oI • ~, . .,..,,:
-:;"': .. , . ~~ . ~:. Y< ...
.:
0.01 0.1 1 0.01
E.l:t!,E. }:,2.1 e nchymaJ
n = 68
0.1
• • .-• •
• • • • •
• • • e ••
• • . ')- .. • • • • .. '"1 •
a .: D. • •• • e •
1 10
IDEAL LUMINAL AREA (mml)
71
Fig. 12B The smooth muscle (SM) relative (rel) variability obtained by dividing the observed by the predicted amount (SM PRED) of smooth muscle takes into account the size of the airways. This smooth muscle variability is plotted against the idealluminal area to represent the distribution of the relative variability in smooth muscle i!' all-sized intra- and extraparenchymal airways.
1
•
IN'tRAPARENCBYMAL
o 0.001
n =381
o
0.01
-0 . -•••• -... , ... o •
o .... •
0.1
o
1 0.01
EXTRAP ~..RENeHYllA.L
0.1
.a = eB
•
o
• • 0.- 0 •• 0
• 0 ct ··0 .: I·~~o
D· O ~. 00 ••
0·0 •
• 10
IDEAL LUMINAL AREA (mm-)
72
Rmax. Large airways (Ibm =2-2.99 mm) were not considpred since two of the animaIs
had no airways in thls category and another two had only one airway. There was no
relationship either between the:. smooth muscle of Is (r=O. !43) or Il (r=O.160)(figufc
14A), or the smooth muscle in the top 25% of intraparenchymal alrways (r=0.224) ~ii1d
Rma}(.
There were no significant relationships betwcen the logECso and the ASM of the
total intraparenchymal airways (r=-0.178) (figure 13B), the smalt (r==-0.270) or medium
(r=-0.280) sized airways, or Is (r=-0.264) or Il (-0.067) (tigure 14B), or the smooth
muscle 111 the upper quartile of the intraparenchymal airways (r=-O.055).
When the intraparenchymal airways were divided into cartilaginous and membranous,
there was no correlation between Rmax and ASM in the cartllaginous (r=0.160) or in
the membranous (r=0.041) group (ilgure 15A). But there was a slgmficant correlation
between the logEC5o and ASM in the cartlhtgmous alrways (r=-0.591, p<O.05). The
negative coefficient of correlation indicates that decreasing values of the 10gEC~{) (which
means increasing sensltivity) .ire associated wlth increasing amounts of ASM. Lastly,
no correlation was found between logECso and ASM in the membranous airways (r=-
0.069) (figure 15B).
3.5.2 Extraparenchymal airways.
The correlation coefficient between Rmax and the ASM of thi~ alrway catcgory was -
0.397 and that between logECso and the ASM was 0.487; neither was signifie:>. 1. (figures
13A and 13B).
73
Fig. 13A The maximal response (Rmax) is plotted against the airway smooth muscle (standardized) for all intraparenchymal (n =361) and extraparenchymal (n =68) airways for the 13 guinea pigs whose lungs were challenged with methacholine for the complete concentration-response curves. The correlations were not significant (p > 0.05).
1
INTRAP ARENCHYMAL EXTRAP ARENCHYMAL
5 r=O.117 r=-O.369
• • .-Irl
" 4 • • P""'4
~ • • • ON 3 • • == El • • • • • • (J 2 '-'
H • • • • as • • • • El 1 ~
0 0.0 0.1 0.2 0.3 0.00 0.05 0.10
.AIRWAY SMOOTH MUSCLE
74
Fi~. 138 The sensitivity (logECso) is plotted against the airway smooth mus.:;Je (standardized) for intra- and extraparenchymal airways for the 13 guinea pigs employed in performing complete concentration-response curves. The correlations were not significant (p > 0.05).
INTRAP ARENCBYlIAL EXTRAPARENCBYMAL
6 r=-O.178 r=O.487
• • ... • 0
• • • • • ~ 2
U •• • • ~ • • 1111 • • • 0 ~ 0 .. • •
-2
• • -4 "------~--~--..... 0.0 0.1 0.2 0.3 0.00 0.05 0.10
AIRWAY SMOOTH MUSCLE
75
Fig. 14A The relationship between maximal response (~x) and airway smooth muscle (standardized) in airways divided according to the median airway in each of 13 guinea pigs. Small = airways less than the median. Large = airways greater than the median. Correlations were not significant (p > 0.05).
'.
~:r .004 a " o 3 =N a ~2 H cd
El ~
1
0 0.0
INTRAP ARENCBYKAL SMALL
r=0.148 • .. .. •
• • • • • •
•
L
0.1 0.2 0.3 0.0
INTRAP ARENCHnIAL lARGE
r= 0.160 • • • • •
• • • .. • •
-1
0.1 0.2 0.3
AIRWAY SYOOTH MUSCLE
76
Fig. 148 The relationship between the sensitivity (logECso) and the airway smooth muscle (standardized) in airways classified according to the median airway in the 13 guinea pigs. Small = airways less than the median. Large = airways greater than the median. Correlations were not significant (p > 0.05).
INTRAPARENCHYYAL SMALt
6 r=-O.264
• 4 • • • •
o 2 • • If) t,) J!I;1 • -- • • 0 0 -~ •
-2
• -4------~--~~ ____ _ 0.0 0.1 0.2 0.3 0.0
INTRAP AREN CBYllAL LARGE
• •
.-
0.1
r="-O.06·1
• • • • • -.
• 0.2
J
0.3
A!RWAY SllOOTH YUSCLE
77
Fig. ISA The relationship between the maximal response (~)() and airway smooth musde (standardized) in membranous and cartilaginous cirways. The corre:lations were not significant. (p > 0.05).
-~
l
5
........ ID
"- 4 .... a "-0
INTRAPARBNCBnIAL MEIlBRANOUS
r=O.041 •
• • • =N 3 • a • • CJ • -- 2 H • • '" s • • ~ 1
o ~--~----~----~ 0.0 0.1 0.2 0.3 0.0
INTRAPARENCBnIAL CARTILAGINOUS
r=O.130
• • • •
• • • •
• • • •
0.1 0.2 0.3
A1RW.AY SMOOTH MUSCLE
78
Fig. 158 The relationship between scnsitivity (logECso) and the airway smooth muscle (~iandardized) in membranous and cartilaginous intraparenchymal airways. î 'here Wéll) a significant correlation in intraparenchymal cartilaginous airways (p < 0.05), but none was found in membranous airways.
'.
INTRAPARENCHYMAL INTRAPARENCIMIAL MEMBRANOUS CARTILAGINOUS
6 r=-O.069 r=-O.591
• • 4 • • • •
; 0 2 • 10 • <.
tJ PQ • .. • • 0
-: r .. ~
-41 • • 1 -L-_--.-J
0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3
AIRWAY SMOOTH MUSCLE
79
Il. Quick-frcezing of guinea pig lungs.
3.6 Methacholine (MCh) challenge.
Table 10 shows the values of lung resistance (RJ for guinea pigs 14 to 19, whose
lungs were frozen at 0, 15, 40, 60, 75 and 100% of their corresponding maximal
resistance, determined from each animal's concentration-response curve. The baseline
resistance was 0.07+0.0l. The maximal response, Rmax, was the highest value
observed in the platea~ of the concentration-response curve. An of the ammals reached
a maximal response following an aerosol of 128 mg/ml, which was the highest
concentration of three aerosols given. The exception was guinea pig 19 whose lung
resistance peaked at 32 mg/ml, and then decreased progressively through 64 and 128
mg/ml of MCh.
There was a 2.2 fold differen~e in the maximal resistance between the least
responsive animal (#3) whose Rmax was 1.41 cmH20/ml/s, and the most responsive
animal (#5) whose Rmax was 3.16 cmH20/ml/s. The values of RL at the time of
freezing ranged between 0.41 in guinea pig 2 and 2.37 in guinea pig 5 (a 5.78-fold).
3.7 Morphometry.
Figures 16A, B, C and D are photomicrographs (xlO, x25, x40) of airways from
quick-frozen lungs. Figure 16A illustrates a constricted airway from a guinea pig whose
lungs were frozen when the pulmonary resistance was at 40% of its Rmax. The
epithclium is highly infolded as a result of the smooth muscle constriction and the airway
lumen is narrowed in contrast to a dilated airway from the control guinea pig (figure
80
00
~ ~
TABLE 10. LUNG RESISTANCE IN QUICK-FROZEN LUNGS (cmH20/ml/s)
1 Guinea Pig Weight %Rmax Aerosol of MCh (mg/ml) Rmax RL%Rmax Number (g) Base 32 64 128
14 331 control 0.01
15 310 15 0.08 1. 70 2.06 2.70 2.70 0.41
16 316 40 0.06 1.22 1.14 1.41 1.41 0.56
17 350 60 0.09 2.54 2.11 2.66 2.66 1. 60
18 402 75 0.10 2.94 2.91 3.16 3.16 2.37
19 400 100 0.07 1. 72 1. 70 1.47 1. 72 1. 72
_L..-..-- - _- -- -- - -_ .. _---
The values of lung resistance represent the average of 5 to 10 breaths and are expressed in cmH20/ml/s. SEM were negligible (see section 2.5.2).
Rmax = the maximal resistar.ce in cmH20/ml/s RL%Rrnax = The absolute resistance in crnH20/ml/s at which the lung was frozen
Fig.16 A. Photomicrograph of an airway from a lung frozen at 40% Rmax. B. Airway from a controllung, Dot expose<! 10 methacholine.
( 82
1
• ~
r' :L 1 ~ ... .... J ... ~
A'': .. t ~ t ~ ,'" ' .(1 \ ~
~ ,"4 JI< ,
Figs. 16C and D Photomicrographs of airways from a lung frozen at 60% Rmax. Note how airways from the same animal may )how different degrees of airway narrowing.
83
16B). In the unconstricted airway the epithelium does not show such infolding. In fact
in figure 16B the airway is almost fully dilated and a nearly circular airway.
FIgures 16C and 16D show all'ways from a lung frozen at 6O%Rmax. The first
airway is so constncted that the airway lumen is practically closed. The second airway
is relatively unconstricted and the lurn~n is relatively patent even though the epithelium
shows sorne infolding. Within the sarne animal, airways exhibited different degrees of
narrowing.
3.8 Distribution of airways sampled.
The distributIon of sizes for airways sarnpled in aU the animaIs (n=6) who se lungs
were frozen with liquid mtrogen is shown in figure 17. Airway size is expressed as the
length of the basement membrane (Ibm). A total of 462 airways were measured, all of
whlch were mtraparenchymal. Also, the number of airways varied among animaIs from
64 ln guinea plg at 15% Rmax to 106 in guinea pig at 40% Rmax. There was an
unequal distnbutlon of alrway sizes with sorne guinea pigs having more large airways
than others. Airway size varied widely from as little as 0.27 mm (Ibm) in guinea pigs
5 to 5,26 mm (Ibm) in guinea pig 6. For airways < 3 mm, the mean airway size did
not vary widely among animais ranging from 1.02+ 0.08 mm (guinea pig 19) to 1.20
+ 0.10 mm (guinea pig 17). The mean value for airways < 3 mm was 1.12 + 0.03
which is almost the same as that obtained for the intraparenchyrnal airways of formalin-
tÏxed lungs (table 11).
The frequency distribution is shown in figure 18 for three size categories, 0-0.99 .. ' j
84
h
Fig. 17 The distribution of intraparenchymal airway:; (n =462) based on the length .)f the basement membrane is plotted for each of the guinea pigs whose lungs were fixed in liquid nitrogen. The number of airways per animal ranged between 64 to 80, and the airway size ranged between 0.27 to 5.26, although most werc less than 2mm. (Compare with fig. 8).
1
Di.tribuUOD of ainrayB Bampled ln tro •• n ,uln... ptl Inn,_
6 -e n:el0R n~.'~ a ~=tJg .......
5 ~
n.::44 a a.=80 Ils ...
,Q
a 4-t)
S .. :
f!I 3 n=>a5 1> : a w Il • Z cZl
lM • 0 • ~
• • 1 ; ... !
ti 1 ! ~ ...l .
! . 0 1 • ..1. -----l
Control 15 4-0% 60" 75" 100%
Gulne. Pi, Number
(
85
TABLE 11. The mean size of. aIl intraparenchymal alrways in quick-fro~en lungs basad O~ tb~ l~nqth of. the basement membran~ iLB~i.
---------
Guinea Pig LBK n
(%Rmax) X ± SEM
C 1.16 + 0.11 80
15 1. 04 ± 0.10 64
40 1.15 + O.OB 106
60 1.20 + 0.10 69
75 1.12 + 0.0'7 65
100 1. 02 + 0.08 78
TOTAL 1.12 ± 0.03 462
---------------------
Values of LBM are expressed as means in mm + SEM. n = number of airways C = control
86
Fig. 18 Frequency distribution of airways according to airway size determined by the length of the base ment membrane. Each dot represents the number of airways in a given animal for a particular size category. The numb'!r in brackets shows when 2 animaIs had the same number of airways. There were 275 airways in the group O-O.99mm (range 36-44), 138 airways in the group 1-1.99mm (range 19-26), and 51 airways in the grovi '2mm (range 6-14).
lrequency distribution 01 alrwaya
ln frosen auinea pic l1U1p
70
• 60
& 50 • • 1 -40 • ~ • 0
... JO G
j 1 20 • •
1 • 10 ~ • J • • • 0-0.98 1-1.ie >2
I..encth 01 buement m.eUlhrme (mm.)
'."
87
mm, 1-1.99 mm and >2 mm in Ibm. There were 275 airways, or 59% of the total, in
the first group, 138 airways, or 30%, in the second, and 51 airways, or Il %, in the last
group. In comparison wlth the formalin-fixed lungs (section 3.3), the distribution of
airway~ was similar for the qUIck-frozen lungs. In both cases, 95 % of the
mtraparenchymal mrways werc in the range < 2 mm in Ibm. Compare figures 17 and
18 with figures 8 and 9.
3.9 Airway narrowing.
3.9.1 Distribution of airway narrowing among all-sized airways.
Alrway narrowmg was expressed by the ratio of the luminal area (LA) to the ideal
lummal area (fLA) (calculauon of ILA is explained in section 2.5.1). In figure 19 data
t'rom a control animal are compared wit.l those from one frozen at 60% of its Rmax.
The LAIlLA is plotted against the length of the epithelium. Assuming that the
unconstricted airway is a perfect circle, a value of 0 on the y-axis represents complete
airway c10sure whercs a value of 1 means that an airway is a perfect circle when
unconslnCled. The control anImai appears to have most airways with lumens that are
approxll11alely 80% of lhat predicted by the ideal luminal area. This is not surprising
smcc lhese Jungs were frozen at Jow transpulmonary pressure. In contrast, the animal
whose Jung was frozen at 60%Rmax shows a heterogeneous distribution of airway
narrowlllg III ail SIZed airways. Heterogeneity is especially noticeable in the small
airways (lep < 2 mm). The same heterogeneous distribution of bronchoconstriction was
obscrved in ail of the Meh exposed guinea pigs.
88
Fig. 19 Distribution of airway narrowing (LA/ILA) as a functlon of airway ~aze glven by the length of the epithelium. Assuming that the unconstncted atrway is a perfect circle, a value of 0 on the y-axis represents complete airway closure whereas a value of one means that an atrway is unconstricted. Here, a control lung is compared with one frozen at 60% of Its Rmax. In the control, LA/ILA ranged From 0.55 to 0.93, 10 contrast to the constricted Jung where LAIlLA ranged From 0.01 to 0.98, showing a heterogenous distribution of constnctlOn.
1
(
h
1.0
0.8
:§ 0.6 ..... ...........
;j 0.4
0.2
GPl (CONTROL)
. . -, . ~. ... ft •
tt· • • • .. ~ ,..-. • • :1 •
0.0 '----4-_-'-----"_~---JL--......I a 1 2 3 4 5 6
GP4 (60% Rmax)
/.. • ,.. • ..
• • • • • •• • •• • •• •• • •• •
• • • . , . • •• •
• ~;"::'L--·--'-_·--II"L--·-'I_-IJ o , 234 5 6
LENGTH OF EPITHELIUM (mm)
89
3.9.2 Distribution of airway narrowing in small, medium, large and extra-large airways.
Table 12 presents the distribution of airway narrowing for ai rway sizc categories 0-
0.99 mm (small), 1-l.99 mm (medIUm), 2-2.99 mm (large), and >3 mm (extra-large)
in Ibm. Values are expressed as the mean LA/ILA (±SEM). The number of airways
in each size class is aIso shown. ln the controllung, LA/ILA was similar among airways
of aIl sizes. In small and medium-sized airways, LA/ILA was almost the samc with 0.76
+ 0.01 and 0.75 + 0.02 respectively. The large and extra-large airways werc slightly
more dilated with relative lumen sizes of 01.<8 + 0.02 and 0.81 + 0.08.
In the constncted lungs there was a progressive increase in airway narrowing from
the small to the larger airways. For instance, in the lung frozen at 6O%Rmax the airway
lumen was 0.51 +0.05 in small airways, 0.23 +0.05 in medium airways, 0.09 + 0.03
in large and 0.04 + 0.02 in extra-large airways. A similar trend was observed in the
other animais.
Sorne of the lungs had no airways in the large size category. For example, the lung
frozen at 15%Rmax had no airways between 2 and 2.99 mm. But the airways > 3 mm
which exhibited ti.e greatest narrowing still allowed for comparison with the smaller
airways, showing the progressive increase in narrowing toward central airways. This
change in airway narrowing was not so drastic in the lung frozen at 40%Rmax, with an
LA/ILA of 0.47 + 0.03 in small and 0.35 + 0.07 in medium size airways. In the large
airways LA/ILA was even slightly hlgher at 0.38 + 0.10. But in contrast to the
previous animaIs, the small airways were more narrowed, and the tendency towards
greater constriction is quite noticeable with an LA/ILA of 0.21 + 0.19 in the extra-large
90
1 .~ ~ .. ~ '\
TABLE 12. Distribution of airway narrowing (LA/ILA) in different sized airways of quick-frozen 1ungs.
GP LBM n LBM n LBM n LBM n 0-0. 99rnrn 1-1. 99mm 2-2.99 mm >3 mm
c 0.76 ± 0.01 44 0.75 ± 0.02 26 0.88 + 0.02 6 0.81 + 0.08 4
15%Rmax 0.63 ± 0.04 39 0.40 ± 0.07 22 0 0.10 ± 0.07 3
\0 40%Rmax 0.47 ± 0.03 67 0.35 ± 0.07 25 0.38 ± 0.10 Il 0.21 ± 0.19 3 ....-
60%Rmax 0.51 ± 0.05 37 0.23 ± 0.05 24 0.09 ± 0.03 5 0.04 ± 0.02 3
75%Rmax 0.39 ± 0.05 36 0.16 ± 0.04 19 0.27 ± 0.11 10 0
100%Rmax 0.67 + 0.04 50 0.38 ± 0.07 22 0.44 ± 0.13 5 0.05 1
Total 0.57 ± 0.06 275 0.38 ± 0.08 138 0.41 + 0.13 37 0.24 + 0.15 14
Values are expressed as means + SEM GP = guinea pig C = control
airways.
The largest dtfference in airway narrowing can be seen in the lung at 75 % Rmax
where LAIILA was 0.39 + 0.05 in small airways and 0.16 ± 0.04 in medium airways,
a 2.44-fold variation. These small airways are the most constricted of all the animaIs.
Surprisingly, the large airways were less narrowed than the medium size airways. There
were no extra-large airways in this animal.
Finally, even in the lung at l00%Rmax, whose level of constriction is uncertain,
there was an increase in airway narrowing from the small airways (LA/ILA =0.67 +0.04)
toward the large (LA/ILA=0.44+0.13) and extra-large (LAIILA =0.05) airways.
3.9.3 Distribution of airway narrowing in small and large airways di'lided according to the median airway.
As explained in section 3.8, there was a somewhat unequal distribution of airway
size with sorne guinea pigs having more large airways than others. For this reason, the
airways were divided according to the median airway in each animal h order to sludy
airway narrowmg ln airways of comparable size. The resuIting size categones arc small,
for airways smaller than the median, and large, for airways larger than the median
airway. In the large airways airway narrowing was greater but not significantly differcnt
(p> 0.05), than in small airways (tigure 20). When airway narrowing is compared
among guinea pIgS, it gradually increases with increasing bronchoconstriction,
partlcuJarly in the large airways (see tables 13 and 14).
92
Fig. 20 Airway narrowing, LA/ILA, is plotted for guinea pigs at different levels of bronchoconstriction. The values shown are means + SEM, for small airways (less than the median) and large airways (larger than the median airway). Note that there was more narrowing in the large airways.
1
"
1.0
0.8
:3 0.6 ~
j 0.4
0.2
0.0--
A1RWAYS: .= SVAJJ,
0= IARGI
c 15~ 40'; 80" 75"
GUINEA PIG
93
~-....."
\0 ~
TABLE 13 Distribution of airway narrowing (LA/ILA) in airways of quick-frozen lungs divided according to the median airway.
GP fi 18 n c. v. IL
Control 0.78 ± 0.01 41 0.12 0.78 + 0.02
15%Rmax 0.66 ± 0.04 32 0.33 0.36 + 0.06
40%Rmax 0.50 ± 0.03 53 0.50 0.34 ± 0.04
60%Rmax 0.56 ± 0.05 34 0.55 0.24 ± C.05
75%Rmax 0.40 + 0.05 33 0.75 0.21 ± 0.04
Values of LA/1LA are expressed as means ± SEM, and have no units 18 = intraparenchymal airways less than the median airway IL = intraparenchymal airways greater than the median airway c.v.= coefficient of variation (x/sd)
n c.v.
41 0.13
32 0.82
53 0.97
35 1.12
32 1.14
yi'-
TABLE 14. The median value and the range of airway narrowing (LA/ILA) in quick-fro~en lungs.
Guin ... ::.. 'nig LA/ILA LA/ILA
(%Rmax) MEDIAN RANGE
C 0.79 0.55 - 0.93
15 0.66 0.03 - 0.88
40 0.44 0.02 - 0.95
60 0.38 0.01 - 0.98
75 0.16 0.02 - 0.92
Values of LA/ILA are expressed as medians and have no units.
C == control
95
3.9.4 Cumulative frequency distributions.
The cumulative frequency distributions are used here to compare the fraction of the
airways that have narrowed to a certain degree, as determined by the ratio of LA to ILA.
Figure 21 A shows the cumulative frequency distribution of ali-sizt'd airways (n =386)
from lungs frozen at 15, 40, 60 and 75 %Rmax. The number of airways as a fraction
of the total is plotted against LA/ILA. In the control lung, 50% of the airways have
their luminal area at about 80% of the ideal luminal area. In contrast, in the lung at
75%Rmax, 50% of the airways are quite narrowed with the lumen occupying less than
20% of the ideal lumen. Thus, with increasing bronchoconstriction a Iarger fraction of
the airways show more narrowing. There was a significant difference between the
control curve and those of the MCh-treated lungs (Kolmogorov-Smimov 2-sample test,
p<O.OI). 'lnere was also a significant difference between the curves at 15 and
40%Rmax (K.S. p<O.05), but no significant difference was found between 40 and 60
%Rmax or between 60 and 75 %Rmax.
A similar picture emerged from cumulative frequency distributions for LA/ILA in
smal1 (Ibm: 0-0.99 mm), and medium (Ibm: 1-l.99 mm) airways. This graph was not
plotted for large airways (Ibm > 2 mm) because the number of airways was too small.
(See figures 21B and 21C). The major difference was that in the medium airways a
larger fraction of airways were more narrowed than in the small airways. This is evident
by a prominent shift upward and to the left of the frequency distributions (figure 21C).
In both groups the curves of constricted lungs were significantly different from that of
the control (K.S. p<O.05), except for the curve at 15%Rmax and the control in the
96
Fig. 21A The cumulative frequency distIibution for aIl sized airways (11 == 386) from lungs fixed in liquid nitrogen is shown for increasing It!vels of airway narrowing (LA/ILA). Each curve represents a guinca pig at control, 15, 40, 60 and 75% of Rmu with 80, 64, 106, 69 and 65 aIrways respectlvely. With increasing degree of bronchoconstriction, a larger fraction of airways showed more narrowing. The control curve was significantly different from aIl the others (p < 0.01).
1
1.0
-ta ~ 0 ~ 0.8 .... 0
~ 0 ;::
0.6 (,)
If l: ...... & 0.4 t ~ .... 0 0.2 . 0
:Ii!:
0.0 1 1
0.0 0.2 ·0.4 0.6 0.8 1.0
Airway NarrowiDi {U/ILA}
97
Fig. 21B The cumulative frequency distribution for increasing levels of airway narrowing (LA/ILA) in small-sized airways (Ibm = 0-0.99mm; n =225). \Vith increasing levels of bronchoconstriction, there IS a larger fraction of airways that show greater airway narrowing. Bach curve represents a guinea pig at control, 15,40, 60, and 75% of Rmax with 44, 39, 67,37 and 36 airways respectively. There is a significant difference between the control and al! the curves (p < 0.05), except for that which corresponds to IS % Rm8'('
1
'c
..
1.0
-:; 0.8 .s -.. 0
~ 0 0.6 ;3 0 • .t: -& 0.4
~ -.. 0 . 0 0.2 ~
0.0 ~ __ -,--__ ~ __ --,-__ ---1 __ --l
0.0 0.2 0.4 0.6 0.8 1.0
Airw." Nurowilac (U/IU)
98
Fig. 21e The cumulative frequency dIstribution for increasing levels of airway narrowing (LA/lLA) in medium-sized airways (Ibm = 1-1.99mm; n = 138). In contrast to the small airways (Ibm = 0-0.99mm) a larger fraction of these airways showed greater airway narrowing (see text). Each curve represents one guinea pig at control, 15, 40, 60, and 75% of Rma'l with 26, 22, 25, 24 and 19 airways respectively. There was a significant difference betwecn the control and all other curves (p < 0.05).
1.0
".... .. ., .s 0.8 .... 0
s:2 0 ;1 CI
0.6
• l: -& 0.4
t ~ .... 0.2 0
• 0 :z:
0.0 0.0 0.2 0.4 0.6 0.8 1.0
Ainray Narrowml (Ll/tLl)
{ 99
small airways.
In figure 22A the median LA/ILA is used to relate airway narrowing deterrnined
morphometrically with the change in pulmonary mechanics expressed as %Rmax. As
the level of bronchoconstriction increased, there was a graduai decrease in the median
lurninal area. Of note is guinea pig 19 which has been included in this graph for
comparison, but il cannot be considered as a reflection of the state of bronchoconstriction
at lOO%Rmax, because its resistance peaked at 32 mg/ml but then decreased through 64
and 128 mg/ml of MCh. In addition, the time period between the application of MCh
aerosol and the fixation with liquid nitrogen may have allowed the constrÎctcd Jung to
recover to an uncertain degree. A similar decrease in airway lumen is seen in figure 228
where the median LA/IL/\ is plotted against the absolute resistance at which the lungs
were frozen, the RL %Rmax (also see table 14). These results are consistent with those
shown in figures 23A and 23B where the mean LA/ILA from small and large airways
are plotted versus the degree of bronchoconstriction. The coefficient of variation of
LA/ILA shown 10 table 13 increases dramatically with increasing degrees of
bronchoconstnction, and correlates weil with %Rmax in small (r=0.98) and large
(r=0.88) airways, indicating that heterogeneity of airway narrowing corrclatcs with
constriction.
3.10 Distribution of airway smooth muscle (ASM).
Figure 24A presents the distribution of airway smooth muscle in mm2 (non
standardized for airway size) as a function of airway size given by the ideal luminal area,
100
Fig. 22A The median LA/ILA has been plotted against the degree of bronchoconstriction, in order to relate airway narrowing determined by morphometry with the change in pulmonary mechanics expressed as % Rmax. There was a graduai decrease in the median luminal area as the level of bronchoconstriction increased.
..... _----, ... ~---------------------------~
-
1.0 r 0.8 CONTROL
j e GP2 (15X Rmu) .....
'" 0.6 :s \',P3 (40" Rmu)
~ • 0.4 • ~ r:aQ GP' (80" Rmu) ::il
0.2 •
GP5 (75" Rmaz)
0.0 0 20 40 60 80 100
LUNG RESISTANCE (%Rmax)
, ..... J.
101
Fig. 228 The median value of airway narrowing (LA/ILA) is plotted against the absolute resistance at which ~.ach lung was frozen (RI %~x). The median luminal area decreases with increasing bronchoconstriction.
1.0
CONTROL ~ 0.8
:s 15~ Rmu r • \ .... t. ......... ::>.6 ~ :s
40% Rmax , ~,
~ i • ~ ~
0.4 • Il
~ 60" Rmu r ~ , ~ ~ l· 0.2 r
75% Rmu ~ • ; ,
: 0.0 " 0 1 2 3 4 ,
~ %Rmax (cmH2O/ml/s)
102
Fig. 23A The relationship between the mean airway narrowing (LAIILA) and the degree of bronchoconstriction expressed hy lung resistancc (% RmIlX) has been plotted for small airways (Le. airways that were smaller than the median airway. See text). The mean luminal area decreased with increasing bronchoconstriction. The values shown are means + SE.
1.0
CONTROL 0.8
t 15% Rmax :s t-004 0.6
40" Rmas ! 60~ Rmax "'-:j ! ~ 0.4 ! 75~ Rmax f:IQ ~
0.2
0.0 '-----L----'-__ ..L.-_---I'--_---'
a 20 40 60 80 1 00
LUNG RESISTANCE (%Rmax)
10.3
Fig. 238 The reJationship between airway narrowing (LAIILA) and the degree of bronchoconstriction expressed by Jung resistance (%~x) bas been plotted for large airways (i.e. airways that were larger than the Median). Here there was a more pronounced increase in airway narrowing with an increase in bronchoconstriction than in smaller airways shown in the previous figure. Values are means + SE.
"
1.0
0.8 CONTROL
:J ~ 0.6 ......... :s 15'; Rmu
~ ! 40~ Rmax
0.4 r:a:l
~ 60% Rmu: ::!1
0.2 ! t 75% Rmax
0.0 0 20 40 60 80 100
LUNG RESISTANCE ("Rmax)
104
Fig. 24A The distribution of airway smooth muscle (ASM) as a function of airway size given by the idea.lluminal area (ILA) for guinea pigs whose lungs were fixed with liquid nitrogen. A total of 95 intraparenchymal airways are shown. Since the smooth ml''ic1e has Dot been standardized for airway size it increases with increasing airway size. (Compare with fig. 12).
0.3
• .--.. 0.2 • 9 ~
::a • rIl • < 0.1
•
0.0 L--__ -D .. ~
0.001 0.01 0.1 1 10
!LA (mml )
,~
105
! "
for four of the guinea pigs whose lungs were fixed in liquid nitrogen. A total of 95
lfitraparenchymal airways are shown. As expected, the smooth muscle increases with
inc.rease in alrway size.
The relative variability ln smooth muscle among all airways is shown in figure 24B
as a functlon of alrway size. This is obtained by dividing the observed by the predicted
smooth muscle (see sectIon 3.4.1). As in formalin-fixed lungs, the distribution of smooth
muscle here IS also heterogeneous (compare with figure 12B).
Wh en the mean amount of ASM, standardized for airway size, is plotted for each
animal (figure 24C), no siglllficant differences appear arnong them. It ranged from 0.15
+ 0.02 to 0.23 + 0.02, a 1.5 fold difference. The mean values of ASM are reported
in table 15. The rnean amount of ASM was 0.18 + 0.02, which is the same as that
rneasured in the intraparenchyrnal airways of formalin-fixed lungs. Also the amount of
ASM in sm ail airways was lower (0.14 + 0.03) but not significantly (p>0.05) different
than that of large airways (0.20 + 0.02).
3.11 Airway narrowing as a function of airway smooth muscle.
To determme if the amount of airway smooth muscle was relate.d to the degree of
airway narrowmg, LA/ILA was plotted against the airway smooth muscle (standardized
for airway size). However, there was no correlation between LA/ILA and ASM (figure
25). In fact, for a given amount of ASM there was a heterogeneous distribution of
airway narrowing. For instance, in the guinea pig at 6O%Rmax, when the amount of
ASM is about l, sorne of the airways appear to be fully dilated near an LA/ILA of 1.0,
106
Fig. 24B The smooth muscle (SM) relative (rel) variability is plotted here against the ideal luminal area. The distribution of smooth muscle appears to be heterogenous for 95 of the airways obtained from the lungs fixed in liquid nitrogen. (Compare with fig. IZB).
(
~ 3
; ~ 2
• ~
t! :II fil 1
0 0.001
INTRA.P ARlNCBYIlAL n=95
• •
... • • -..
• • •• • • • •• • • .. :-..... ., . •• • • ••• , 1-4\, • • ·fII •
0.01 0.1
mEAL LUIONAL ARIA (mm·)
107
•
• •
• 1
..... 0 00
~ :,
TABLE 15. Amount of airway smooth muscle (ASM) in intraparenchymal airways of quickfrozen lungs, divided according to median airway.
GP (%Rmax) Is n IL n TOTAL
Control 0.18 ± 0.01 14 0.17 ± 0.01 20 0.18 + 0.01
15 0.20 ± 0.03 9 0.26 ± 0.09 11 0.23 + 0.02
60 0.08 + 0.01 10 0.17 ± 0.02 17 0.14 ± 0.02
75 0.11 + 0.01 8 0.20 + 0.04 6 0.15 ± 0.02
TOTAL 0.14 ± 0.03 41 0.20 ± 0.02 54 0.18 ± 0.02
n
34
20
27
14
95
Values of ASM are expressed as means ± SEM and have no units. I8 = intraparenchymal airways less than the median airway
n = number of airwayso
Il = intraparenchymal airways greater than the median airway GP = guinea pig
Fig.24C The amount of smooth muscle (standardized for airway size) is shown for four guinea pigs whose lungs were frozen at 15 %, 60% and 75 % of ~x. C is the control animal. The differences in smooth muscle among animals were not significant (p > 0.05). Values are expressed as mean + SEM. n = number of airways.
"
0.3
n::20
~ t.) fil n=3.j. p ::il 0.2
n=14-
= n=27 E-4 0 0 )1 fil
~ 0.1
~ A:: t-4
<
0.0 '--------' c 15% 60% 75%
GTJINEA PIG
109
Fig. 25 Airway narrowing (LAIILA) is plotted against the amount of smooth muscle standardized for airway size. There was a heterogenous distribution of airway narrowing for a given amount of airway smooth muscle.
•
\ ,
CONTROL 15% Rmu
1.0 1.0
• • • • • , • • 0.8 Cc· ...
0.4 • • l • • • • • • • • • 1;; • •• • •
1 • •• • •
0.8 • • Il • •
0.4
• • 0.2
0.2 •
( • • 0.0 •
, 0.0 f 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1
-L-.. __ J i 0.2 0.3 0.4 0.5
f f
~ j •
~ 607. Rmu
75r. Rmu: 1.0 1.0 •
~
• • ••• • O.I! •
0.8 • • • •
0.8 0.8
• • •
0.4 • • 0.4
• • 0.2 0.2
• • • • • ,. • '-. • • 0.0 • 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5
AIRlVAY SMOOTH MUSCLE ~.
110
(
while others appear to be completely closed with an LA/ILA near O.
3.12 Smooth muscle shortening (SM shortening).
3.12.1 Distribution of smooth muscle shortening.
The smooth muscle shortening, expressed as a per cent, has been plotted against the
length of the epithelium, to describe its distribution across airways of different sizes
(figure 26). (Calculation of %SM shortening can be found in section 2.5.2). In the
control, the %SM shortening was on the average about 20%, and it was constant across
di fferent-sized airways. In contrast, the bronchoconstricted an.l;nals exhibited
heterogeneity in the %SM shortening than the control. For example the lung at
15%Rmax had airways with less than 10 %SM shortening, and the lung at 60%Rmax had
airways with even less %SM shortening near O. In the lung at 75%Rmax the smallest
airways were not sampled (i.e. airways sample-d were between 0.2 and 3.0 mm in lep),
but heterogeneity ln shortening was observed for the range sampled. Also, in contrast
to the control, the constricted airways showed considerably more SM shortening, which
approached 70 to 80 % .
3.12.2 Smooth muscle shortening as a function of ASM.
The %SM shortening is plotted against the airway smooth muscle (standardized for
airway size) in figure 27. In the control animal, the smooth muscle shortens by about
20 %, regardless of the amount of smooth muscle. Surprisingly, in the constricted lungs
the SM shortening varies from nearly 0 to about 80% of the predicted value for the
unçonstricted aIrway. The distribution of SM shortening as a function of ASM appears
III
Fig. 26 The smooth muscle (SM) shortening, expressed as per cent, is plotted agamst the length of the epithelium (LEP) at different levels of bronchoconstrictlon. In the control animal the SM shortens by about 20% in all sized airways, whereas in the constricted lungs, the SM shortening var. '5 from near 0 to almost 80% of the predicted value. Note that there was no relationship between the % SM shortening and LEP.
, .1
CONTROL 15" Rmaz
100 100
• 10 •
• 40 •
• • • "'~ ..•. •• •
20 20 •• • • • · ._, . " 0 0 --l. ! - --.J
0 2 3 4 5 0 2 3 " !
~ Z ~
Z rzl E-t g::
80';; Rmas 0 757. Rmu = 100 100 rn ::il rn 80 10
~ • • • • ••
eo ... • 60 •• • • • •
• 40 • • •
• •• • • 20 • 20 • • • • ... , ..
a lit • 0 0 1 2 3 4 5 0 2 3 4 5
LENGTH OF EPITHELIUM
( 112
Fig. 27 The smooth muscle (SM) shortening expressed as per cent, is plotted agamst the amount of smooth muscle (standardized for airway size). In the control the SM shortens by about 20% regardless of the amount of smooth muscle, whereas in the constricted lung, the SM shortening varies from about 0 to nearly 80% of the predicted value. Note that there was no relationship between the % SM shortening and the amount of smooth muscle.
1" ;.
't
CONTROL 15~ Rmax
• \JO 100
ao ao
• 50 •
• 40 •
e • •
~ 20 ee-Wr ••
20 • • .. Z . .... • • • ..... • • • Z 0 0 ~ 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5
~ g: 0
60~ Rm8.% = 75~ Rmax CIl
)1 100
CIl ~ 80
• • • • • •
60 •• • 80 .,. • •
• •
40 • 40 • • •
•• • • 20 20 • • • • • . ,. • • • • 0 .. -----J 0 --L...-. 1 ...J
0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5
AIRWAY SAlOOm MUSCLE
J
113
to be heterogeneous. There was no relationship between the %SM shortening and the
amount of smooth muscle.
114
1)
2)
3)
4)
5)
( '\
SUMMARY OF RFSULTS
The quantity of ASM does not appear to determine differences in maximal bronchoconstriction among normal guinea pigs; the lack of a correlation between responsiveness and amount of ASM may be explained by the heterogenous distribution of bronchoconstriction among the airways studied or the modality of challenge.
The sensitivity to MCh appears to be related to differences in the amount of ASM in intraparenchymal cartilaginous airways.
Variability in the ECso may also reflect differences in airway cross-sectional area.
Lung resistance appears to be a good measure of constriction since the morphometric measure of airway narrowing correlated weIl with resistance.
The heterogeneity of airway narrowing does not appear to be determined by differences in ASM.
115
Chapter 4
DISCUSSION
4.1 Methacholine (MCh) responsiveness.
The results of this study showed substantial variability in the position of the
concentration-response curve to inhaled methacholine, both in relation to the x-axis, the
maximal response, and to the y-axis, the sensitivity. These findings are consistent with
previous studies (44-50,86) which also found a large variabllity in the responses of
guinea pigs. The variability in the sensitivity recorded in this study was a 254 fold
difference between the least and the most sensitive animal, which is larger than the 100
fold range reported by Douglas (49) in spontaneously breathing unancsthctized guinca
pigs, and the 22 fold variation reported by Hulbert (86) in mechanically ventilatcd guinea
pigs.
It has previously been shown that the sensitivity of airway smooth muscle to inhaled
bronchoconstrictive agents shows variability from animal to animal within ail spccies that
have so far been studied. Bai et al. (10) found among cats a 23 fold variation in
sensitivity to methacholine, as recorded by the EC200R, (the effective concentration of
agonist required to increase the resistance by 200% from baseline). Snapper and
colleagues (164) performed histamme dose-response curves in dogs and descnbed a
greater than 40 fold difference in the EC200Rt . In an extensive study done in humans,
Townley et al. (183) reported variability in sensitivity to aerosohzed methachohne among
116
Chapter4
DISCUSSION
4.1 Methacholine (MCh) responsiveness.
The results of this study showed substantial variability in the position of the
concentration-response curve to inhaled methacholine, both in relation to the x-axis, the
maximal response, and to the y-axis, the sensitivity. These findings are consistent with
previous studies (44-50,86) which also found a large variability in the responses of
guinea pigs. The variability in the sensitivity recorded in this study was a 254 fold
difference between the least and the most sensitive animal, which is larger than the lOO
fold range reported by Douglas (49) in spontaneously breathing unanesthetized guinea
pigs, and the 22 fold variation reported by Hulbert (86) in mechanically ventilated guinea
pigs.
It has previously been shown that the sensitivity of airway smooth muscle to inhaled
bronchoconstrictive agents shows variability from animal to animal within ail species that
have so far been studied. Bai et a1. (10) found among cats a 23 fold variation in
sensilivity lo methacholine, as recorded by the EC200RL (the effective concentration of
agonist required to increase the resistance by 200% from baseline). Snapper and
colleagues (164) performed histamine dose-response curves in dogs and described a
greater than 40 fold difference in the EC200RL. In an extensive study done in humans,
Townley et al. (183) reported variability in sensitivity to aerosolized methacholine among
116
1 patients with allergie rhinitis, current asymptomatic asthmatics, former asthmatics, and
nonatopic controls. MCh challenge (5 mg/ml) was performed ln 98 subjects and
sensitivity to MCh was defined in thelr study by a greatcr than 20% decrcase in the
forced expiratory volume in 1 second, or FEYI. In normals, the percent dccrcasc III
FEV, ranged From 1 to 26% in normaIs, 1 to 41 % In subjects with allergie rhinitis, 1 to
43% in former asthmatics, and 20 to 64% in current asthmatics. ln anothcr study also
done in humans but using the effective dose of inhaled carbachol required to cause a 25%
decrease in conductance (ED2SGaw), Orehek et al. (141) reported a 20 fold variation in
asthmatics and a 10 fold variatIOn in normals. The dlfference between normals and
asthmatics was 60 fold. Habib et al. (67) also recorded vanation in airway responses to
inhaled histamine in a normal population of humans. They measured pulmonary
resistance and the maximum expiratory tlow rate at an absolute lung volume
correspondmg to 40% of the control VItal capacity, oblamed during forced expIration
• • From tldal end inspiratIOn, or Vmax-tüp. Responses measurcd by RI and Ymax41~1 vaned
by a factor of 32 and 38 respectively.
The demonstration of plateaus on the concentration-responst! curves IS also consIstent
with previous investigations (119,174,193), which found that normal subjects show a
plateau on the dose-reponse curve to histamine (119,193) and methacholine (174). The
concentration-response curves reported here, showed variabl!Jty in the maximal responsc.
This variability, however, was much less than that of the sensltlvlty. In fact, there was
only a 3.6 fold variation in Rmax among the guinea pigs challengcd to methacholine.
There is litt]e evidence in the literature on the characterization of the complete dosc-
117
response curves, probably due to the difficulty in achieving plateaus in human subjects,
especia11y m asthmatlcs. In experiments done JO humans it is partÎCularly difficult to
assess vanablhty of maximal response since many asthmatics may not reach a plateau.
Woolcock et al. (193), for instance, attempted to characterize the shape of dose-response
curves by performing bronchial challenge to inhaled histamine in current asthmatics, mild
asthmatics, and normal subjects. The current asthrnatics in their study failed to reach a
plateau despite a 60% fall in FEY l, although in the mild asthmattcs and the normals the
challenges were contmued until a plateau was reached. The measurements of the plateau,
given by the percent fall in FEV l ranged from 15 to 45%, a 3 fold difference, among the
normal subjects. The mild asthmatics had values of 43 and 34%. Even though using the
FEV l the variability may appear small, other investigators using different techniques have
also reported a slInilar range of measurements. Michoud and colleagues (119) performed
dosc-response curves to histamine mhalation in normal subjects and asthmatics,
measuring lung reslstance, and observed that 6 of 17 subjects tended to reach a plateau.
In thcse subjects the maximal response varied by a factor of 2.5. A plateau can also be
observed ln 8 of the subjects studied by Habib et al. (67), with a 3.5 fold variation in the
maximal responses.
The sources of the variations in the maximal response and the sensitivity are as yet
incompletely charactenzed. lndividual differences in sensitivity may be related to
dlffcrenccs ln alrway structure, breathing pattern, metabolism of the agonist, or other
factors wlllch determine how much agonist reaches airway smooth muscle
(67,92,124,164). Interammal variability in the maximal response cou Id be related to
118
L
l
~.'
differences in the strength or the amount of smooth muscle, anered )ength-Ienslon
relationships in smooth muscle, changes in the loads on the smooth muscle, and other
changes in the structural components of the airway wall (8.16,45,92,124,128). The fact
that there is not a relationship belween the maximal rcponse and the sensltlvity is
consistent with the notion that the factors which determine these lwo parameters may be
different (45,124). In the CUITent study, guinea pigs with very similar maximal
responses, such as #1 and #9, had extreme values in sensitivlty (0.96 versus 22.63
mg/ml), and guinea pigs with a1most the same sensitivity, such as #1 and #2, had very
different maximal responses (1.61 versus 3.6 cmH20/mlls). Thus, guinea #1 was very
sensitive but not very responsive.
The notion that sensitivity is determined by factors that do not neccssarily alter
maximal narrowing is suggested by studies that have lookcd at the Importance of changes
in aerosol depositlon (1,4,101,162,163.152,176,195,196), changes In the functlon of the
airway eplthelium (15,23.59,65,71,72,128), and changes in ccll-to-cell commul1lcatlon
(2,38,95,171). It has been shown that the sensitlvity may bc affected by deposltlon of
inhaled aerosol, depending particularly on the breathing pattern (124,152,162, ) 63) and
the airway geometry (l, 195,196). ChangIng the ventilatory pattern dunng aeroso)
inhalation could result in preferential central depositlon (152) with high tlows, or
peripheral deposition with low flows. Of course, changes in the breathing pattern are
not an issue here sll1ce aH animaIs received the same pattern of ventilation.
Differences in airway geometr'/ under ic' .;ntical breathing conditions rnay account for
sorne of the vanablhty in the sensitivity recorded. The concept of dysanapsis, the
119
{
, ... difference in the relative proportion of the parenchyma and the airways, has been
propo~.ed to explaJO dlfferences in maxImum flow rates among normal subjects (81). The
relauve proportions of parenchyma and airways vary from individual to individual;
certain subjects have relatively small airways for a given volume of Jung tissue. That
a similar phenomenon occurs in guinea pigs is supported by the fact that the mean size
of the intraparenchymal airways was significantly different from 1nimal te animal. In
fact, the cross-sectlonal areas suggest that there is a relationship between the airway size
and the aerosol deposition in the guinea pig. Animais with narrower airways tended to
be more sensItive to MCh. The variation in airway size did not appear to determine
differences JO the maxImal response. To date there is no complete model of the guinea
pig lung that would allow for comparisons with the data presented here. However, most
of the currently studied models, derived from human lungs, at least demonstrate that
different branching structures and airway dimensions probably occur in different subjects
(70,140,195,196).
Differences in airway dimensions could also alter aerosol deposition (1). For
instance, longer airways would be expected to show more deposition toward distal
airways where tlow velocity is less (see section 1.5). In contrast, smaller airways wou Id
show more central depositlOTl. The total deposition is not significantly affected by Jung
size (88). Central deposition resulting from decrease ln cross-section al area has been
shown In both normal subjects in whom bronchoconstriction has been induced (176) and
in bronchoconstricted asthmatics (lOI), as weIl as in dogs challenged with MCh (160).
Il makes sense that JO a 1 ung with narrcwer airways, inhaled MCh would be deposited
120
1 more centrally and result in higher sensitivity to MCh. Since the longitudinal dimensions
were not measured 10 this study, it is not possible to relate the length of the airways to
measures of airway responsiveness. ft is unclear to what extent particle size wou Id
be important in causing variability among animais. With the method used ln the
experiments of this thesis, the size of particles dehvered presumably ranged from 0.5 to
10 u and 85% were 4.5 to 6.0 u whlch means that most particles were probably
deposited in the central airways and a small percentage reached the peripheral airways
where depositlon occurs with particles 2 to 4 u (181).
Another factor which may have been a source of variability tn sensitlvlty IS the
permeability of the epithelium. If the epithelium is damaged It means the loss of the
physical barrier to constrictive inhaled agonists, and the loss of relaxing factors derivcd
from the epnhehum (15,23.59,65,71). Hlstologicai studlcs fom asthmatlc airways have
demonstrated denudation of the epithelium (23,:.; l). But in the current study therc was
no evidence of epithelial damage on hght microscopy, so this factor IS unlikcly to account
for variations in sensitivity.
4.2 Variability in airway smooth muscle.
The central hypothesis in this study was that the variability in maximal
responsiveness is determined by differences in airway smooth muscle. It was surprising
that there was no correlation between the maximal response and the amount of alrway
smooth muscle. From the analysis of the dose-response curve by Moreno and colleagues
(124), it was predicted that changes in maximal resistance would be determined by
121
.
changes in the quantity of smooth muscle. In their paper, these authors provide a strong
theoretical basls for considering the smooth muscle :m important mechanical factor in
causing alrways responsiveness; too much smooth muscle potentially should result in
excessive shortening, airway narrowing, and hyperresponsiveness. Various
morphologicaJ studies have shown that an increase in airway smooth muscle occurs in
the airways of human asthmatics (74,83,85,167). Other studies that have looked at the
physiological responses of ASM have shown that ASM from subjects with asthma (158)
or obstructive Jung disease (40) who have airway hyperresponsiveness in vivo has
increased strength in vitro, and has a greater maximal response to contractile agonists
(8). Il is uncertain if an increase in strength is related to an increase in the amount of
ASM. The data presented in the current study would argue that a larger amount of
smooth muscle does not result in increased maximal responses. Still, the notion that the
maximal response should be affeeted by differences in the amount of airway smooth
muscle is suggested by experiments that have studied the effeet of changes in lung
volume on lung resistance and the load to shortening imposed by the elastic properties
of the parenchyma (16,45,96,174). In normal subjects the maximal response cao be
affected considerably by changing lung recoil by the induction of experimental
emphysema (16), or by changmg lung volume (45). Therefore the balance of forces
between the contractmg smooth muscle and the elastic properties of the parenchyma
seems to be critical in determining the extent of airway narrowing. But in
hyperresponslve subjects increases in lung volume have been shown to result in smaller
changes in the maximallevel of constriction (96), and this effeet JYI",~ 'Oe attributed to a
122
1 greater mass of ASM that cao withstand the increasing afterload imposed by lung stretch.
It is a1so possible that lung elastlcity may be different. This has not been mvestlgated
in great detai! but early papers have described loss of lung clastic recoil in certain
asthmatic subjects (64).
One possible explanation for the absence of a relationship betwecn responsiveness
and ASM is that the airways that contribute most to resistance may not be the ones Ihal
were sampled and measured morphometrically. With methachol ine, con striction has becn
shown to oecur from the first to the sixth generatlOn (the trachea bemg gencration 0, and
the first two extraparenchymal) of airways, at least In the dog (160). In the cllrrent stlldy
the extraparenchymal airways measured were the trachea and the first and second
generation airways; one group of intraparenchymal alrways includcd airways as far as
the third and fourth generations. So these airways should have provlded a major portIOn
of lung resistance. Still, it is uncertain to what extcnt the intraparcnchymal airways from
the mldsagittal sections contributed to reslstance; Il is not known to what gencration thcy
belong.
An alternate explanation is that the inter-animal variability in the amounl of ASM in
extraparenchymal airways \>:as not large enough for a notlceable signal to appear among
animaIs. In fact, the ASM of extraparenchymal alrways comprised only bctwecn 10 to
30% of the total amount of ASM. Presumably, sigmficant changes may have bcen
recorded with larger values of ASM. Indeed, significant inter-animal differenccs ln ASM
existed only for intraparenchymal alrways, partlcularly thosc obtamed from midsagittal
sectionli. These possessed 70-90% of the total ASM but theIr contribution to lung
123
t:'. resistance is likely to have been small. '. Presumabl y there are no changes in cross-sectional area due to changes in length of
the airways. Kenyon et al. found that airway length reduction h insignificant compared
to the extent of parenchymal tethering of the airway, indicating that the airways are stiff
longitudmally (97).
Several studles have shown that airway sensitivity is determined by both genetic and
environ mental factors (17,57,75-77,180). Eidelman et al. (57) have demonstrated that
differences in alrway smooth muscle appear to account, at least in part, for the
differences ln responsiveness among highly inbred rat strains. The presence of strain
related responsiveness in rats strongly supports the idea that genetic factors are
Important. Therefore, it is possible that genetically determined differences in ASM may
account for differences in sensitivity in the guinea pig. Whether the maximal response
is genetlcally determined has not been studied. Takino et al. (180) performed pedigree
breeding for 5 years in 2 lines of guinea pigs with bronchi sensitive and insensitive to
ACh, and conc1uded that sensitivity of the bronchial wall is inborn. Likewise, these
authors did not look at maxImal responses. There is also convincing evidence that
envirQnmental factors may modulate responsiveness. Hyperresponsiveness can be
mduced by exposure to antlgen (16,34), cigarette smoke (78), viral infections (58), and
recently repeated exposure to inhaled ACh in guinea pigs (134). The common change
in the first three of the above studies was airway inflammation; with ACh the major
finding was an lncrease in the airway wall thickness. and an increase in ASM in
peripheral airways but not in the large cartilaginous airways. But these environ mental
124
~-------------------------
{ , Jo
factors are not likely to be important for guinea pigs in the current study since they were
kept in comparable environmental conditions.
4.3. Variability in airway narrowing.
One of the major findings in this study was the substantial varia"ility in airway
narrowing among airways of equivalent size. This heterogencous distribution of
bronchoconstriction may partly explain the lack of a correlation between responsiveness
and the amount of airway smooth muscle. Also the phYSlOlogical measurements arc
made with the basic assumption that the lung is a linear one-corn part ment modeI, and that
consequently, it constricts homogeneously. In correlating the ASM wlth the maximal
reslstance, it is assumed that the physiological measuremcnt of alrway narrowmg is the
end result of the contribution to airway narrowing by the ASM from a representative
sample of airways measured by morphometry. This may not be the case, but it is
unlikely to be a problem in the present study where hundreds of airways werc measured.
As explained earlier, the pattern of aerosol deposition IS unlikely to be uniform. The
results on the distribution of airway narrowing show that the larger airways constnct to
a larger degree than the smaller airw~ys. Once proximal airways constrict, they may
block further entry of aerosol toward distal airways. This pomt of constnctlon may vary
from airway to airway within an ammal and may be dlfferent from one anImal to the
other. Such a phenomenon would explain why airways of the same slze narrow to
different degrees.
Pulmonary resistance is at best expected to provlde only an indirect measure of
.'
125
airway calibre. The other factor which could contribute to the measurement of Jung
resistance is tissue VI seance. Lung resistance is made up of tissue viscance (Ytis) and
airways resistance. Tissue viscance represents the pressure changes across the alveoli
that are in phase with airflow and independent of flow rate (87,107,115). Il is unknown
what the contribution of Vtis is to Jung resistance in the guinea pig, but it is likely to be
substantial. In rabbits Romero and Ludwig (151) obtained maximal responses to MCh
and found that Vtis accounted for the major proportion of RL both under control
conditions and after MCh-induced constriction. Considerable variations in Vtis are likely
to be caused by differences in the tidal volume and frequency used (87). Larger tidal
volumes and low frequencies result in higher Ytis (61,87). In the current study these
parameters were close to physiological values and were held constant throughout the
experiment in ail animaIs, so Vtis is unlikely to have been a major cause of variability
among guinea pigs. Desptte the above arguments there was a good correlation between
the level of bronchoconstrictlOn and the degree of airway narrowing among animais
whose lungs were frozen with liquid nitrogen. This shows that there is a good agreement
between the morphometric measure of constriction and the physiological parameter that
reflects alrway narrowing, the pulmonary resistance.
ln summary, there is no simple relationship between ASM and airway responsiveness.
The role of ASM remains to be cIarified. About all that is certain is that excessive
spasm of ASM can cause problems, which in humans means asthma. It is evident that
ASM has an active role in controlling regional and generalized distribution of air to the
lungs. But in Iight of the functlOnal consequences of ASM contraction which are
126
presumably to minimize airway resistance and ventilatory dead space, it IS intriguing to
find differences in the amount of ASM within a species, wllhout more clear cut
cOITesponding differences m responslveness. Contrary to the basic hypolhcsis of the
current study, the quantity of ASM does not appear to delermine differences in maximal
bronchoconstriction, or in the distribution of airway narrowing ln the guinea pig.
Instead, variations in the amount of ASM may be related to differences in the sensltivity.
This finding and the lack of a correlation between responsiveness and the amount of
ASM may be explained by the heterogeneous distribution of bronchoconstriction among
the airways studled or the modahty of challenge. Nonetheless, the present model is a
useful way to further explore the mechanical factors involvcd in alrway responsivcness.
The characterization of the smooth muscle distribution throughout the tracheobronchlal
tTee of the guinea pig, as well as the determination of complete concentration-response
curves to Meh should prove useful to fllrther understanding the fllnctional and structural
role of airway smooth muscle within this species. It would be mtcrestmg to relate the
findings in guinea plgS to human phYSIOlogy and, perhaps, to the human dlsease of
asthma. But comparisons are difficult since comparable data in humans are not available
and there is no true animal equivalent of asthma.
127
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