Structural failures of the blood-gas barrier and the epithelial-epithelial cell connections in the different vascular regions of the lung of the domestic fowl, Gallus gallus variant domesticus, at rest and during exercise.

1.6 MB·English

by  Maina, John N.

Research Article 267
Structural failures of the blood–gas barrier and the
epithelial–epithelial cell connections in the different
vascular regions of the lung of the domestic fowl,
Gallus gallus variant domesticus, at rest and during
exercise
John N. Maina1,* and Sikiru A. Jimoh2,`
1DepartmentofZoology,UniversityofJohannesburg,AucklandPark2006,Johannesburg,SouthAfrica
2SchoolofAnatomicalSciences,UniversityoftheWitwatersrand,Braamfontein2000,Johannesburg,SouthAfrica
*Authorforcorrespondence([email protected])
`Presentaddress:DepartmentofBiochemistry,FacultyofHealthSciences,UniversityofCapeTown,Observatory7935,CapeTown,SouthAfrica
BiologyOpen2,267–276
doi:10.1242/bio.20133608
Received12thNovember2012
Accepted11thDecember2012
Summary
Structural failure of blood–gas barrier (BGB) and workloadsmayhavearisenfromhemodynamicchangesthat
epithelial–epithelial cell connections (EECCs) in different initially ensued from exudation of blood plasma and then
vascular regions of the exchange tissue of the lung was flow of blood into the air capillaries on failure of the BGB.
n
studiedinrestedandexercisedchickens.Thenumberofred TherelativedifferencesinthenBGBBsandthenEECCBsin
e
p blood cells (nRBCs) was counted and protein concentration the different vascular regions of the lung were ascribed to
O
(PC) measured after lavaging the respiratory system, and diameters of the branches and their points of origin and
y blood was sampled to determine the blood lactate levels anglesofbifurcationfromthepulmonaryartery.Presenceof
g
o (BLLs). The numbers of complete BGB breaks (nBGBBs) RBCs in the air capillaries of the lungs of rested chickens
ol
and those of the EECCs (nEECCBs) were counted in the showed that failure of the BGB commonly occurs even in
Bi
differentvascularterritoriesofthelung.ThenRBCsandthe healthy and unstressed birds. Rapid repair and/or defense
PCs increased with increasing exercise intensities but the responses,whichwereobserved,mayexplainhowbirdscope
rateofincreasedecreasedathigherworkloads.Fromrestto with mechanical injuries of the BGB.
the fastest experimental treadmill speed of 2.95 m.sec21,
BLLs increased 4-fold. In all cases, the nEECCBs exceeded (cid:2)2013.PublishedbyTheCompanyofBiologistsLtd.Thisis
those of the BGB, showing that structurally the BGB is an Open Access article distributed under the terms of the
relatively weaker than the EECC. The increase in the CreativeCommonsAttributionNon-CommercialShareAlike
numberofbreakswithincreasingexercisecanbe attributed License (http://creativecommons.org/licenses/by-nc-sa/3.0).
to increase in the pulmonary capillary blood pressure
(PCBP) from faster heart rates and higher cardiac outputs, Keywords:Birds,Exercise,Rest,Chicken,Lung,Blood–gasbarrier,
while the leveling out of the measurements made at higher Structural failure
Introduction withoxygen.Comparedtomammals,thearterialbloodpressures
‘‘Like most machines, components of the body eventually fail. inbirdsaremuchhigher(Spector,1956;SpeckmannandRinger,
Diseasestates,mechanicalinsultornormalwearmayinitiatethe 1963; Erickson, 1994; Seymour and Blaylock, 2000;
process of tissue degradation.’’ (Li et al., 2010) Lichtenberger, 2005). For example, compared to that of
The air capillaries and the blood capillaries of the exchange 95 mmHg (12.64 kPa) in a mammal like the cynomologus
tissueoftheavianlunghavebeenreportedtobehavelike‘rigid’ monkey, Macaca fascularis, (body mass 4.60 kg), the turkey,
tubes (Macklem et al., 1979; Powell et al., 1985; West et al., Meleagris gallopavo, (4.77 kg) has a higher systolic blood
2007;Watsonetal.,2008).Likemammals,birdsareendothermic pressure of 150 mmHg (19.95 kPa) (Seymour and Blaylock,
homeotherms; they operate at relatively higher body 2000). Turkeys have exceptionally high systolic blood pressures
temperatures (40–42˚C) (Aschoff and Pohl, 1970). Oxygen is of as much as 400 mmHg (53.3kPa) (Beller et al., 2004). In
acquired by a structurally complex and functionally efficient ratites (mainlyostrich, emu,andrhea),sudden deathshavebeen
respiratory system, the lung air sac system (e.g. Powell and attributedtoaorticrupture(MitchinsonandKeymer,1977)(2008
Scheid, 1989; Maina, 2005). To support an energetic lifestyle, factsheet ‘Aortic ruptures’ by Hunter et al.: http://www.
large hearts (Hartman, 1955) with large cardiac outputs (Grubb, agbiosecurity.ca/healthybirds/Factsheets/Disease/AorticRupture.
1983;Bishop,1997;SeymourandBlaylock,2000)supplytissues pdf). Termed ‘dissecting aneurism’ or ‘turkey heart attack’, fast
Structural failure in the chicken lung 268
growing,healthyturkeys(mostlymalesofbetween7to24weeks tolerate for the set exercise program; they had to be physically
of age) die suddenly of bleeding after aortic rupture which is nudged to keep running and complete the time.
caused by high blood pressure (Collins, 1971; Neumann and
Ungar,1973;Kristaetal.,1986;vanVeen,1999).Afterautopsy Blood lactatelevels
of a visibly (externally) healthy cardinal, Cardinalis cardinalis, For the exercised chickens, the pre-exercise BLL values were
which died after a short territorial spat, a 7-mm hole (probably comparablewhilethepost-exerciseonesincreasedgraduallywith
causedbyacutebloodpressuresurgeduringtheexcitement)was increasing workload (Fig. 1). In the rested chickens, the
reportedintheventricleoftheheart(Erickson,1994).Birdslike difference between the first BLL measurement (1.3960.01s.d.
the fast growing broiler chickens and turkeys frequently mmol.L21) and the second one (1.2760.09s.d. mmol.L21),
experience hypertensive problems that lead to high myocardial measurements taken 10 minutes apart, was not statistically
stress, resulting in complications such as aortic aneurysm and significant (0.5.P.0.1). The small but consistent difference
ascites(SpeckmannandRinger, 1963; Kristaet al., 1970;Julian could have ensued from incidental distress of the chickens prior
and Wilson, 1986; Currie, 1999; Issac et al., 2010). In to taking the first measurement. The same can explain the small
hypertensive turkeys, respectively, Simpson, and Krista et al. differences between the resting groups of birds in the different
(Simpson,1978;Kristaetal.,1967)reportedmortalityratesfrom exercise regimens. Between rest and running speed of
aorticruptureof 20% and43.7%. Itisperplexing that withtheir 0.66 m.sec21, post-exercise BLL rose from 1.2960.09s.d.
relativelyhighsystolicandarterialbloodpressures,birdspossess mmol.L21 to 3.1860.04s.d. mmol.L21, a 2.5-fold increase;
a blood–gas barrier (BGB) which is ,36thinner than those of between speeds of 1.97 and 2.95 m.sec.21, the increase of BLL
from 4.0360.18s.d. to 5.1460.61s.d. mmol.L21 was not
mammalsofequivalentbodymass(Gehretal.,1981;Mainaand
significant (P.0.5) for a 1.56 increase in the running speed
King, 1982; Maina et al., 1989; Maina and West, 2005; Maina,
(Fig. 1). This shows that under the exercise regimen, BLL
2005).
approachedorreachedthehighestphysiologicallytolerablelevel
This studyinvestigated the effect of exercise on the structural
and that metabolically, the birds were pushed close to their
integrityoftheBGBandtheepithelial–epithelialcellconnections
exercise endurance limit.
(EECCs) in the different regions of the exchange tissue of the
lung of the domestic fowl (chicken), Gallus gallus variant
Redblood cellsand proteinconcentrations inlavaged fluid
n domesticus,whicharesuppliedbythefourmainbranchesofthe
e pulmonary artery (PA). Changes of blood lactate levels (BLLs), In both rested and exercised chickens, red blood cells (RBCs)
p were observed in the recovered fluid (Fig. 2); they were
O numbers of red blood cells (nRBCs) and protein concentrations
indicative of BGB failure. The nRBCs increased up to the
gy (PCs) in lavaged fluid were assessed and complete blood–gas treadmill speed of 1.97 m.sec21 after which it leveled out
barrier breaks (BGBBs) and epithelial–epithelial cell connection
o (Fig. 2). While between resting and the treadmill belt speed of
Biol burseeadkasb(bErEevCiCatBiosn)swiesregicvoeunnitnedT.aAblleist1offodreqfuinicitkiornefoefrecnocmem. only 0.98 m.s21thenRBCsincreasedfrom36105646104s.d.percm3
to56105666103s.d.percm3,afactorialincreaseof1.7,between
the speeds of 1.97 (5.96105626104s.d. per cm3) and that of
Results 2.95 m.s21 (6.46105666103s.d. per cm3) the increase was only
At the slowest treadmill speed of 0.66 m.sec21, some of the 1.1.Atthetreadmillspeedof1.97 m.s21,thechickensstartedto
chickens tried to jump off the treadmill belt but were prevented
struggle to keep running, particularly after 7 minutes on the
by a transparent perspex sheet, which was placed well above it.
treadmill. In the fluid recovered from the respiratory system of
At speeds of 2.53 and 2.95 m.sec21, after 7 minutes of running,
the chickens, PC increased gradually from rest to different
the chickens were visibly exhausted; they had to struggle to
exercise intensities (Fig. 3). Between a lower level of exercise
complete the set exercise time of 10 minutes. The fastest
and a higher one, the greatest increase occurred between rest
treadmill speed of the experiment (2.95 m.sec21) appeared to (8.8560.14s.d.mg.ml21andthetreadmillspeedof1.97 m.sec21
be the maximum exercise intensity that the chickens could (34.0360.23s.d. mg.ml21), a factorial increase of ,4. Showing
thatexercisewasaffectingtheminthesameway,thechangesin
the nRBCs (Fig. 2) matched those of PC (Fig. 3).
Table 1. Definition of abbreviations commonly used in
the text. Blood–gas barrierbreaks andepithelial–epithelial cell
connection breaksin different vascularregionsofthe lung
Abbreviation Definition
In rested and exercised chickens, BGBBs and EECCBs were
BGB Blood–GasBarrier observedinthedifferentregionsofthelungsuppliedbythefour
BGBBs Blood–GasBarrierBreaks
mainbranchesofthePA.Inallcases,thenEECCBsexceededthe
BLL BloodLactateLevel
BGBBs (Fig. 4). The differences between the nBGBBs and the
EECCBs Epithelial–EpithelialCellConnectionBreaks
EECCs Epithelial–EpithelialCellConnections nEECCBsinthedifferentvascularterritoriesofthelung(Figs 5,
nBGBBs numberofBlood–GasBarrierBreaks 6) and the differences between the nEECCBs and those of the
nEECCBs numberofEpithelial–EpithelialCellConnectionBreaks BGB in a particular vascular region of the lung (Fig. 7) were
nRBCs numberofRedBloodCells
expressed as percentages of the total in order to reflect the
PA PulmonaryArtery
PC ProteinConcentration relativenumbersofstructuralfailuresintheseparts.Fig. 5shows
PCBP PulmonaryCapillaryBloodPressure thatintherestedchickens,comparedtootherregions,theregion
RBCs RedBloodCells suppliedbythecaudomedialbranchhadmostfailures;nobreaks
VV_O2 Oxygenconsumption occurredintheregionsuppliedbythecranialbranch;thenumber
VV_O MaximumOxygenconsumption
2max. ofbreaksintheareassuppliedbytheaccessoryandcaudolateral
Structural failure in the chicken lung 269
Fig.1. Bloodlactatelevels(BLLs)ofchickensatrest
andafterexercise.Thepost-exercisevaluesincreasedwith
increasingexerciseintensitybuttherateofincrease
decreasedwithincreasingworkload.Inrestedchickens,the
differencesbetweentheBLLmaybeexplainedbysome
distresspriortotakingthefirstBLLreading.
branches was comparable. At the treadmill speed of accessory and caudomedial branches of the PA had the greatest
0.66 m.sec21, the region supplied by the cranial branch had the and the cranial and caudolateral branches the lowest numbersof
leastnBGBBswhilethatsuppliedbythecaudomedialbranchhad breaks. Fig. 7 shows the relative differences between the
n
the greatest number; the number of breaks in areas supplied by nEECCBs and the nBGBBs in the different vascular regions of
e
p the accessory and caudolateral branches was comparable. At the the lung. It shows the most common type of break and how the
O
treadmill speeds of 2.53 and 2.95 m.sec21, the nBGBBs did not numbers of breaks in the four regions of the exchange tissue
y
g show much variation in the four regions. Fig. 6 shows that in compare at rest and during different exercise intensities. In the
o rested chickens, the greatest nEECCBs occurred in the region restedchickens,intheareasuppliedbythecranialbranchofthe
ol
Bi suppliedbytheaccessorybranchofthePAwhilethelowestwas PA, the EECCBs were the only breaks which were observed
in the region supplied by the caudolateral one; the values in the while the areas supplied by the other branches showed marked
regions supplied by the cranial and caudomedial branches were differencesoftherelativenumbersofEECCBstoBGBBs;inthe
similar. At treadmill speeds of 0.66 and 0.98 m.sec21, the area supplied by the caudomedial branch, the EECCBs
nEECCBs in the regions supplied by the accessory and contributed 55% of the total number of breaks in the vascular
caudomedial branches were comparable and more than in the region and for those supplied by the accessory and caudolateral
regionssuppliedbythecranialandcaudolateralbranches.Atthe branches the values were 77% and 78% respectively. At
treadmill speed of 1.97 m.sec21, the nEECCBs showed modest 0.66 m.sec21 treadmill speed, the percentage differences were
variation. At treadmill speeds of 2.53 and 2.95 m.sec21, the lowerthanthoseinrestedchickens,withtheareasuppliedbythe
Fig.2. Changesofthenumberofredbloodcells
(nRBCs)inthefluidcollectedfromtherespiratory
systematrestandafterexercise.Thegreatestincreaseof
thenRBCsoccurredbetweenthetreadmillexercisespeeds
of0.66and1.97m.sec21whiletherateofincreaseofthe
nRBCsaboutleveledoffathigherworkloads.
Structural failure in the chicken lung 270
Fig.3. Changesofproteinconcentration(PC)inthe
fluidcollectedfromtherespiratorysystemofthe
chickenlungatrestandafterexercise.Thegreatest
increasesofPCoccurredbetweenthetreadmillexercise
speedsof0.66and1.97m.sec21whiletherateofincrease
ofPCdecreasedathigherworkloads.
cranialbranchofthePAshowingthegreatestrelativecontribution et al., 1999). In a horse, Thomas and Fregin observed that at
of the EECCBs and the caudomedial one the lowest. At maximumexerciseonatreadmill,strokevolumeincreasedbyas
0.98 m.sec21, the values were about equal while at the treadmill much41%abovetherestingvalueandcardiacoutputrose6-fold
speedof1.97 m.sec21andhigher,therelativeproportionsbetween (Thomas and Fregin, 1981). Oxygen consumption (VV_O ) of
2
theEECCBsandtheBGBBsgenerallydecreased. animalsrunningonatreadmillincreasedlinearlyasafunctionof
speed (e.g. Gleeson and Baldwin, 1981; Taylor et al., 1981). In
Morphologiesof theblood–gas barrierbreaks andthe the marabou stork, Leptoptilos crumeniferus, Bamford and
n epithelial–epithelial cellconnection breaks Maloiy reported a direct relationship between VV_O and heart
e 2
p The profuse interdigitation of the air capillaries and the blood rate forbirdsrunningontreadmill (BamfordandMaloiy,1980),
O
capillaries in the exchange tissue of the lung is shown in while large increases in the respiratory rate and heart rate were
y Fig. 8A,B. The EECCs are for the most part thin cellular reportedbyButleretal.,Butler,andPetersetal.inbirdsflyingin
g
o extensions that separate the air capillaries while connecting the a wind tunnel (Butler et al., 1977; Butler, 1991; Peters et al.,
ol blood capillaries (Fig. 8A–C). The failure of the EECCs started 2005).Eliassenreportedthatpulsepressurealmostdoubledfrom
Bi with small perforations (Fig. 8D) which joined into large slits rest to flight in gulls (Eliassen, 1963), while Hart and Roy
(Fig. 8E,F), while for the BGB, early injury was heralded by recorded3to4timesincreaseinheartrateovertherestinglevel
measurableincreaseofthenRBCsandthePCinthelavagefluid in exercising pigeon (Hart and Roy, 1966). Increase of BLL in
(Figs 2,3);atlowerworkloads,linearbreaksoccurred(Fig. 8G) blood during exercise has been shown to occur in three phases
andathigherones,thebloodcapillarywalltoreopen(Fig. 8H,I). (Roston et al., 1987; Cabrera and Chizeck, 1996): in the first
phase which corresponds to less than 60% of maximum VV_O
2
Discussion (VV_O ) and occurs during mild to moderate exercise, the rate
2max.
Runningonatreadmillisahighlyenergeticformofexercisethat of glycolysis increases several times without significant BLL
occasionslargeenergyexpenditure(Rostonetal.,1987;Cabrera increase;inthesecondphasewhichoccursduringheavyexercise
Fig.4. Changesintheaveragenumbersofblood–gas
barrierbreaks(nBGBBs)andepithelial–epithelialcell
connectionbreaks(nEECCBs)inlungsofrestedand
exercisedchickens.Thebreaksincreasedwithincreasing
exerciseintensity,withthedifferencesbetweenthetwo
typesofbreaksdecreasingathigherworkload.Bothatrest
andexercise,thenEECCBsexceededthenBGBBs.
Structural failure in the chicken lung 271
Fig.5. Relative(%)differencesofthenumbersof
blood–gasbarrierbreaks(nBGBBs)inthedifferent
regionsofthelungsuppliedbythefourmainbranches
ofthepulmonaryarteryinrestedandexercised
chickens.Intherestedchickens,thenumberofbreaksin
thecaudomedialbranchwasthehighestandnobreaks
occurredintheareasuppliedbythecranialbranch;
betweentherestedchickensandthoserunatatreadmill
speedof0.66m.sec21,thenumbersofbreaksintheregions
suppliedbytheaccessorybranchofthePAwere
comparable.Inrunningchickens,theareassuppliedbythe
caudomedialandaccessorybranchesgenerallyhadthe
highestnumberofBGBbreaks.
equivalentto60to80%ofVV_O ,BLLrisestoahighersteady- occurred.Asimilarprocesswasobservedinthedoglungswhich
2max.
state level above the resting one, and; during the third phase were subjected to high transient vascular pressure (Maron et al.,
which occurs when VV_O is greater than 80%, anaerobic 2001). Presence of RBCs on the respiratory surface of normal
2
glycolysis augments the energy derived from aerobic (healthy) birds has been previously reported, e.g. in rock dove,
production, causing progressive increase of BLL (Cabrera et Columba livia, (Maina and Cowley, 1998) and the chicken
al., 1999). Because lactate acid level serves as a diagnostically (Nganpiep and Maina, 2002; Kiama et al., 2008). As confirmed
importantoxidizablesubstrateduringexercise,ithasbeenusedto here, moderate inconsequential structural failure of the BGB
access, plan, and predict training and performance endurance seems to be a common occurrence even in lungs of resting
n programs (Cabrera et al., 1999). (unstressed) birds. Compared to mammals, birds have higher
e
p In this study, BLLs rose 4-fold between rested chickens and arterialbloodpressures(SeymourandBlaylock,2000)andmuch
O thoserunatthefastesttreadmillspeedof2.95 m.sec21.Thepost- largerhearts(e.g.Hartman,1955;SeymourandBlaylock,2000)
y exercise BLLs should have been consistent with the exercise whichgeneratelargecardiacoutputs(e.g.Grubb,1982).Presence
g
o intensities since the chickens were not trained to run before the of white blood cells and fibrinous strands within minutes of the
ol experiments. It cannot, however, be totally ruled out that the experimentshowedexistenceofanefficientcellulardefenseand/
Bi chickens may have suffered some degree of stress prior to and or repair mechanism. It may explain how birds contend with
during the experiments, even though every care was taken to BGBBs that appear to occur commonly in their lungs. The
minimize or eliminate it. The recurrent though insignificant production of the fibrillary structures which may plug BGB
differencebetweenthefirstandthesecondBLLmeasurementsin breaksmaybeinitiatedbyactivationofsmallGTPasesRhoand
rested birds can be attributed to instinctive stress that may have Cdc42 that are known to stimulate actin filament synthesis
caused the BLL to remain elevated beyond a 10-minute time (Wood et al., 2002). In the mammalian lung, repair of the
interval. The difference between the BLL in the rested chickens alveolar epithelial cells after pressure was increased to injurious
and those subjected to the slowest exercise treatment levels and then lowered to non-injurious ones was reported by
(0.66 m.sec21) can be explained by delay between lactate acid Vlahakis et al. (Vlahakis et al., 2002). In this study, at higher
build-up and release of lactate dehydrogenase. workloads,thechickensmayhavestruggledtocompletethe10-
The initial increase of PC in the recovered fluid may have minute exercise program due to build-up of lactic acid in the
resulted from increased leakiness of the BGB before failure tissues, especially in the leg muscles and occurrence of a
Fig.6. Relative(%)differencesofthenumbersof
epithelial–epithelialcellconnectionbreaks(nEECCBs)
inthedifferentvascularregionsofthelungsuppliedby
thefourmainbranchesofthepulmonaryartery(PA)in
restedandexercisedchickens.Inrestedchickens,the
regionssuppliedbytheaccessoryandcaudolateral
branchesofthePAshowedthehighestandthelowest
numbersofbreaksrespectivelywhilethenumbersofbreaks
werecomparableintheregionssuppliedbythecranialand
caudomedialbranches.Atspeedsof0.66and0.98m.sec21,
thenumbersofbreaksintheregionssuppliedbythe
accessoryandcaudomedialbranchesofthePAwere
comparableandgreaterthanvaluesfromtheregions
suppliedbythecranialandcaudolateralbranchesofthePA.
Generally,inexercisingbirds,theareassuppliedbythe
accessoryandcaudomedialbrancheshadthegreatest
numberofbreaks.
Structural failure in the chicken lung 272
Fig.7. Relative(%)differencesbetweenthenumbersof
epithelial–epithelialcellconnectionbreaks(nEECCBs)
andthenumbersofblood–gasbarrierbreaks(nBGBBs)
indifferentvascularregionsofrestedandexercised
chickenlungs.Fortherestedchickens,theregionsupplied
bythecranialbranch,onlyEECCBsoccurredwhileata
treadmillspeedof0.98m.sec21,therelativeproportionsof
theEECCBswereequivalent.Ostensiblydueto
hemodynamicchangesthatoccurredafterfailureofthe
BGBandflowofbloodplasmaandthenbloodintotheair
capillaries,therelationshipbetweenthenEECCBsandthe
nBGBBsdecreasedfromrestedtoexercisedchickens.In
restedchickens,theEECCsweremorehighlysusceptible
tofailurethantheBGB.
‘respiratoryblock’frominitialpresenceofbloodplasmaandthen separatingtheaircapillariesandconnectingthebloodcapillaries
of blood in the air capillaries. West and Mathieu-Costello (Fig. 8A–C).Theincreaseintherelativedifferencesbetweenthe
observed that stress failure of the BGB is an important but nEECCBs and the nBGBBs at higher workloads may have been
overlooked factor that limits maximum exercise (West and causedbyhemodynamicchangeswhichoccurredafterfailureof
Mathieu-Costello, 1995). theBGB,initiallyleadingtoexudationofbloodplasmaandthen
n Increase inthepulmonary capillarybloodpressure(PCBP)is streaming of blood into the air capillaries. It is plausible that
e
p probably the foremost direct challenge to the mechanical presence of blood in the air capillaries externally stabilized the
O
integrity of the blood capillary wall, especially the BGB, and BGB while accelerating failure of the EECCs. Interestingly, in
y indirectly the EECCs. In racing horses, PCBP rose to chickens infused with 2,4 dinitrophenol (DNP), which caused a
g
o 100 mmHg (13.33 kPa) (Jones et al., 1992). While such high 4-fold increase in cardiac output, for unclear reasons, while the
ol pressure may not have been reached in exercised chickens, pulmonary arterial pressure rose steadily (as cardiac output
Bi
BGBBs which were physically similar to those reported in the increased),theincreaseinthearterialpressurewasevenlessthan
mammalian lung by West et al. (West et al., 1991) were that in a mammal, where recruitment and distension in the
observed. It remains to be determined whether under similar pulmonary capillary system occurs (West et al., 2010).
levels of PCBP and states of BGB thickness the avian BGB is In concurrence with the physics of fluid flow, the point of
stronger than the mammalian one. Bird lungs have BGBs that origin, the angle of bifurcation, and the diameters of the main
are,3timesthinnerthanthoseofmammals(Gehretal.,1981; branchesofthePAshouldprofoundlydeterminethedistribution
Maina, 1989; Maina et al., 1989; West, 2009). For that, the of blood todifferentregionsofthe lung.Fromthepatternof its
avianlungshouldbemoresusceptibletopulmonarymechanical branching reported by Abdalla (Abdalla, 1989) and the
injuries and diseases. After comparing the strengths (stress diameters and angles of bifurcation of the four branches
tolerance) of the blood capillaries in the rabbit lungs, the dog (unpublished observations), more blood should flow into the
lungs, and the horse lungs, Birks et al. observed that stress caudomedialbranchbecauseitisthewidest andthemostdirect
failuremaycorrelatewiththethicknessoftheBGB(Birksetal., continuationofthePAwhilethehighestresistanceshouldoccur
1994), a property consistent with Laplace’s relationship which in the accessory branch because it is the first and the narrowest
states that wall stress is proportional to capillary radius but branch that originates from the PA. Measurements made on
inversely proportional to wall thickness. We have demonstrated latex casts of the lung of the domestic fowl (unpublished
collagen fibers in the basement membrane of the BGB and the observations) showed that the angles of bifurcation of the
EECCs of the lung of the chicken (Maina et al., 2010) and accessory, the cranial, the caudomedial, and the caudolateral
immunolocalized type-IV collagen (Jimoh and Maina, 2013), a branches from the PA were respectively 44˚, 56˚, 17˚, and 90˚
protein family of triple helical isoforms that form strong two- while the diameters were 0.8, 1.5, 2.2, and 1.4 mm. In the
dimensional planar network of fibers (Timpl, 1989; Hudson et chicken lung, no anastomoses occur between the arterial
al.,1993;West,2009),intheexchangetissueof thelungof the branches of the PA (Abdalla and King, 1976). Therefore, with
domesticfowl.ThestrengthoftheBGBofthemammalianlung certainty,theBGBBsandtheEECCBscountedinthisstudycan
has been attributed to presence of type-IV collagen in the be associated with the individual branches of the PA. The
basement membrane (Crouch et al., 1997; West and Mathieu- greatest nBGBBs and nEECCBs occurred in the regions of the
Costello, 1999; Maina and West, 2005; West, 2009). lung supplied by the caudomedial and accessory branches in
The greater nEECCBs compared to those BGB, points out to accordance with respectively the expected greater blood flow
the fact that the former sites are structurally weaker than the and resistance (pressure) in the two branches. Pulmonary blood
latter;theEECCsaretheareasoftheexchangetissueoftheavian flow redistribution was reported in the lung of the panting
lung where thin strands of epithelial cells lie back-to-back, ostrich, Struthio camelus, (Jones, 1982).
Structural failure in the chicken lung 273
n
e
p
O
y
g
o
ol
Bi
Fig.8. (A)Photomicrographoftheexchangetissueofthechickenlungonwhichthenumbersofcompleteblood–gasbarrierbreaks(BGBBs)(dashedsquares)and
epithelial–epithelialcellconnectionbreaks(EECCBs)(circles)werecounted.AC,aircapillaries;BC,bloodcapillaries;asterisks,epithelialcells;stars,endothelial
cells;RBC,redbloodcells;WBC,whitebloodcells.Scalebar:20mm.(B)Transmissionelectronmicrographshowingthecomponentsoftheexchangetissue:air
capillaries(AC),bloodcapillaries(BC),redbloodcells(RBC),epithelial–epithelialcellconnections(circles),barriersseparatingbloodcapillaries(squares),blood–
gasbarriers(arrows),andepithelialcellnucleus(asterisk).Scalebar:20mm.(C)Scanningelectronmicrographshowingepithelial–epithelialcellconnections
(encircledareas).BC,bloodcapillaries.Scalebar:15mm.(D)Scanningelectronmicrographclose-upofperforationsofanepithelial–epithelialcellconnection
(asterisks).BC,bloodcapillary.Scalebar:25mm.(E)Scanningelectronmicrographshowinganepithelial–epithelialcellconnection(areabetweencontinuouslines)
withalargefailuresite(asterisk),smallerholes(arrows)abouttomergewithit,andthedirectionoffailureshownbythedashedline.BC,bloodcapillaries.Scalebar:
30mm.(F)Transmissionelectronmicrographshowinganepithelial–epithelialcellconnectionbreak(boxedarea).AC,aircapillary;BC,bloodcapillary;arrows,
blood–gasbarrier.Scalebar:10mm.(G)Scanningelectronmicrographshowingatransverselinearfracturesite(dashedline)ofabloodcapillary(BC)wall.Scale
bar:25mm.(H)Scanningelectronmicrographshowingareawheretheblood–gasbarrierhasrippedoffathighworkload(star).RBC,redbloodcell;asterisks,
fibrinousstrandsclosetoandoverthefailuresite.Scalebar:10mm.(I)Transmissionelectronmicrographshowingablood–gasbarrierrupturesite(star).BC,blood
capillary;AC,aircapillary;RBC,redbloodcell;WBC,whitebloodcellwithfilopodia(arrows)attachingontotheendothelialcellofthebloodcapillary;Ep,
epithelialcellnucleus.Scalebar:20mm.
Conclusion explain how birds cope with modest BGBBs. The occurrence of
This study corroborates earlier investigations by Maina and more EECCBs (compared to the BGBBs) showed that as would
Cowley, Nganpiep and Maina, and Kiama et al. (Maina and be expected from their morphologies (Maina and King, 1982;
Cowley, 1998; Nganpiep and Maina, 2002; Kiama et al., 2008) Maina and West, 2005), the EECCs are weaker than the BGBs.
that structural failure of the BGB is common even in resting The relative numbers of BGBBs and EECCBs in the different
(unstressed) and normal (healthy) birds. The high arterial blood partsofthelung,whicharesuppliedwithbloodbythefourmain
pressuresinbirds(e.g.SeymourandBlaylock,2000)andsystolic branchesofthePA,correlatewiththevolumesofblooddelivered
pressures that range from 108 to 220 mmHg (14.4–29.3 kPa) to these regions. Identifying how and under what conditions
(LumeijandRitchie,1994),mayleadtohighPCBPs,explaining BGBBs occur in the avian lung should help in formulation of
the occurrence BGBBs even in inactive/unstressed birds. husbandry practices that lower incidences of deaths from such
Presence of white blood cells and prompt deposition of fibrin- injuries. For birds bred for human consumption, husbandry
like material at the failure sites showed existence of an efficient methodsthatminimizestressmayhelpreducelosesandtherefore
cellulardefenseand/orrepairmechanismofinjuredBGB.Itmay improve commercial productivity.
Structural failure in the chicken lung 274
Materials and Methods immersedinthesamefixative,andstoredat4˚Cbeforesamplingandprocessing
formicroscopicanalysisandexamination.
Experimentalanimals
The University of the Witwatersrand’s Animal Ethics Screening Committee
grantedapprovalofthisstudy(ClearanceNumber2007/53/01).Maturefreerange Experimentsonexercisedchickens
mixedbreedofdomesticchickenswhichweighedbetween1.4and2.8kgwere ACollinstreadmillmachineH(UpfrontMerchants,Boulder,CO,USA)powered
obtainedfromareliablesupplier.Theywerekeptinananimalholdingunitfor3 byasingleelectricmotorwithavariablespeedofbetween0.5and3.0m.sec21,
weekstoacclimatize to theindoor conditions,become accustomedto handling, maximuminclinationangleof5˚andtotalrunningbeltlengthof150cmwasused.
andfortheirhealthtobeproperlyassessed.Thetemperatureinthewell-ventilated Priortotheexperiments,thechickenswereallowedtofamiliarizethemselveswith
roominwhichtheywerekeptwasmaintainedat22˚C,thebirdswereexposedto thesurroundingsandbecomeaccustomedtothenoisemadebythemachine;they
12-hourlightand12-hourdarknesscycles,theywerefedacommercialfoodration wereplacedclosetoitfor1houreverymorningfor2weeks.Everyothercarewas
and occasional green vegetables, and water was provided ad libitum. The taken to reduce stress that could increase heart rate or respiratory rate.
experimentalanimalsweredividedintotwogroups;thefirstgroupcomprisedof Measurementsoftheseparameterswereavoidedinordernottoexcitethebirds.
restedchickenswhilethoseinthesecondgroupweresubjectedtofiveexercise Duringtheexperiment,thechickenswerequietlyapproachedandgentlyplaced
regimens.Eightchickenswereusedforeachtest;forthe6experimentalset-ups(1 legsfirstonthetreadmillwhilethemachinewasrunningattheslowestspeedand
restingand5exercisetreatments),atotalof48birdswereused,and;inthefour zero inclination. When the chicken started walking, the speed of the belt was
experimentalformulations(BLLmeasurement,countingofRBCs,determination slowly increased to the desired exercise level. After 10minutes of running, a
of PC, and counting of BGBBs and EECCBs), 192 chickens were used. The sampleofbloodwasimmediatelytakenandtheBLLmeasured.Pulmonarylavage
microscopic morphologies of the BGBBs and the EECCBs were studied by wasperformedandthenRBCsandthePCdeterminedintherecoveredfluid.Inthe
transmissionelectronmicroscopesandscanningelectronmicroscopes. sameexerciseprotocol,thechickenswerefirstinjectedwithheparinthroughthe
leftbrachialveinandafter10minutesofcirculationkilledbyinjectingthemwith
EuthanaseH. The coelomic cavity was opened by a ventral approach, the
Pulmonarylavage
pericardialsacincised,andtheadiposetissuearoundtherootofthepulmonary
The chickens were killed by injection with 5ml of thiopentone sodium
(200mg.cm23) (EuthanaseH) into the right brachial vein and the trachea trunkcleareduptowherethebloodvesselbifurcatedintoleftandrightpulmonary
arteries.Incisionsweremadethroughtherightventricleandalsoacrossthetipof
exteriorized and cannulated. PBS (pH 7.4) which was warmed to 40˚C was
theleftventricletoaccesstheircavities.Theinflowtubewasinsertedthroughthe
instilledintotherespiratorysystemfromaheightof30cmabovethesternum.
slitintherightventricleandpusheduptothepulmonarytrunkandtheoutflow
When it stopped flowing, the trachea was ligated and the fluid left in the
tubewasinsertedthroughtheopeningattheapexoftheheartandpushedpastthe
respiratorysystemfor10minutesbeforecollectingitbygravity,i.e.byholding
atrioventicularvalveintotheleftatrium.Thepulmonarycirculationwasflushed
thebird’sheaddownbyitslegs.
withwarmPBSandthelungsfixedasintherestedbirds.
Countingoftheredbloodcells
Samplingandprocessingoflungtissuesforlightmicroscopy,
Ahemocytometerwith Neubauer markingsand a special coverslip wasused to
transmissionelectronmicroscopyandscanningelectron
counttheRBCs.Countingwasdoneonfivesquaresatatotalmagnificationof
n 4006,usinga106eyepieceanda406objective.Doublecountingwasavoidedby microscopy
e
p applyinganexclusionrule:cellswhichoverlappedthetopandtherightboundary Transverseincisionsofthefixedlungweremadealongthecostalsulciandthe
O ofthecountingareawereincludedwhilethosethattouchedthebottomandleft acquiredslicescutinhalves.Anacetatepaperwithaquadraticsquarelatticegrid
boundarywereexcluded.ThenRBCswascalculatedfromthevolumeofthefluid ofatotalareaof6mm66mmdividedinto1mm61mmnumberedsquareswas
y
g underthecoverslipinthefivesquaresthatwerecounted. placedonthesurfaceofthecranialaspectoftheslice.Fourrandomnumberswere
o generated using a random number generator (free software) and samples taken
ol from areas corresponding to the numbers. Tissue samples were taken from the
Determinationofproteinconcentration
Bi regionsperfusedbythefourmainbranchesofthepulmonaryartery(PA),namely
TheLowrymethodofPCdetermination(Lowryetal.,1951)wasused.Triplicates the cranial, the accessory, the caudomedial and the caudolateral branches. The
of 50ml each of serially diluted working solution of BSA were put in 1mm3 samples taken wereprocessed forlightmicroscopy(LM), transmission electron
Eppendorftubes.Thecollectedlavagefluidwasalsopreparedintriplicate.Two microscopy (TEM) and scanning electron microscopy (SEM) by the standard
hundredmlofreagentAwasaddedtothecontentsoftheEppendorftubeswhich laboratorytechniques.Briefly,forLMandTEM,thesampleswereembeddedin
wereleftonamechanicalshakerfor10minutesatroomtemperature(22˚C).Fifty epoxy resin, semithin sections contrasted with toluidine blue, and for TEM
ml of reagent B was added to the content of each tube and shaken for another ultrathinsectionswerecontrastedwithleadnitrateanduranylacetateandviewed
30minutes. Absorbance of each of the solutions in a microwell was read at on a JEOL100S microscope. Samples for SEM were dehydrated, sputter-coated
690nmwavelengthusingLabsystemsMultiskanAscent(AmershamPharmacia, withgold–palladiumcomplex,andviewedonaJEOL840microscope.
Buckinghamshire, UK) spectrophotometer. The absorbance values of the BSA
standardswereplottedagainstconcentrationstoconstructastandardcurve.The
PC in the lavage fluid was calculated using the derived mathematical equation Determiningnumbersofblood–gasbarrierbreaks(BGBBs)and
relatingtheabsorbanceoftheBSAstandardtotheconcentrationobtainedfromthe epithelial–epithelialcellconnectionbreaks(EECCBs)
graph. The BGBBs and the EECCBs in the vascular regions supplied by the PA were
counted on semithin toluidine-blue stained sections at a final magnification of
Experimentsonrestedchickens 61000,usinga106eyepieceanda1006oilobjective(Fig.8A).Theanalysiswas
madeoneightrandomlyselectedmicroscopicfieldspersection.Thesectionswere
Toavoidagitatingthebirdsbeforethestartoftheexperiment,chickenswerekept
randomly selected from the different vascular regions of the lung. The average
in isolation (in a cage) for 2hours. Calming the chickens by sedation was
nBGBBswascalculatedbyaddingthetotalnumberofbreaksinthevascularregions
considered but not performed due to possibility of introducing experimental
ofthelunganddividingitbythenumberof100mm2countingfields;thesamewas
‘noise’ that would have been difficult to set apart, especially for the exercised
donefortheEECCBs.Therelative(%)proportionsoftheBGBBsandtheEECCBs
birds.Aftercalmingdown,achickenwasgentlyremovedfromthecageandadrop
inthevascularregionsoflungwerecalculatedforcomparisons.
ofbloodtakenbyprickingthecombwithalancet.Threemeasurementsofblood
lactic acid level (BLL) were made with Lactate PlusH (Nova Biomedical,
Waltham, MA, USA) hand-held machine. Next, the chicken was injected with Statisticalevaluation
2.5cm3heparin(1000IU)throughtheleftbrachialvein.Following10minutesof A nested analysis of covariance (ANCOVA) was used to test the differences
itscirculation,thebirdwaskilledbyinjectionof5mlofthiopentonesodiuminto betweensomeoftheparameters(Littelletal.,1991),withthestatisticallevelof
therightbrachialvein.Therespiratorysystemwastheninstilledwithwarm(40˚C) significancesetatP,0.05.
PBSfromaheightof30cmabovethesternum.ThenRBCswerecountedandthe
PCsdeterminedinthefluidrecoveredfromtherespiratorysystem.Afterflushing
Acknowledgements
thelungswithwarmPBS(pH7.4;temperature40˚C)for2to3minutes,theywere
fixed by perfusing them with 2.5% glutaraldehyde (pH of 7.4; osmolarity of This work was funded by the National Research Foundation (NRF)
350mOsm.L21) at an inflow pressure of 12cmHO (8.8mmHg51.2kPa); the ofSouthAfrica.WearegratefultothetechnicalstaffoftheSchool
2
outflow tube was placed at the level of the sternum 7cmH2O ofAnatomicalSciencesoftheUniversityofWitwatersrandfortheir
(5.3mmHg50.7kPa) [In a pilot study, it was determined that an inflow immense assistance; to Mr C. van der Merwe of University of
pressure of 12cmHO (1.2kPa59mmHg) and an outflow one of 8cmHO
2 2 Pretoria for assistance with scanning electron microscopy; Dr V.
(0.8kPa56mmHg) caused minimal or no BGB and EECCs failures]. The
progressofintravascularperfusionwasassessedfromtheblanching(whitening)of Meskiani for training (S.A.J.) in techniques of immunoelectron
thelungduetoejectionofbloodfromit.Thelungswereremovedfromthethorax, microscopy; staff of the Central Animal Service (CAS) Unit of the
Structural failure in the chicken lung 275
University of the Witwatersrand for their help with care of the Krista,L.M.,Waibel,P.E.,Shoffner,R.N.andSautter,J.H.(1970).Astudyof
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The authors havenocompetinginterests todeclare. andhypotensiveturkeys.Br.Poult.Sci.27,207-213.
Li,J.,Connell,S.andShi,R.(2010).Biomimeticarchitecturesfortissueengineering.
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