1^%ÜR Z. Säugetierkunde63(1998)220-227 ZEITSCHRIFT © SAUGETIERKUNDE 1998GustavFischer INTERNATIONALJOURNAL OF MAMMALIAN BIOLOGY Size-independent distribution ofbronchial cartilage in four species of myomorph rodents ByK.-P. Valerius InstitutfürAnatomieundZellbiologie,Justus-Liebig-Universität, dessen, Germany ReceiptofMs. 19. 08. 1997 AcceptanceofMs. 14. 11. 1997 Abstract Thesize-independentdistributionofbronchialcartilageintheconductivebronchialtreeoffourspecies ofmyomorph rodents different in body weight was determined by the inspection oftranslucent carti- lage-stained whole-lung-specimens in comparison to bronchial casts. The lungs of the harvest mouse. Micromys minutus, body weight 5-7g, the laboratory house mouse, Mus musculus, body weight 35- 45g, the laboratory brown rat, Rattus norvegicus, body weight 200-400g, and the African giant pouchedrat, Cricetomysganibianus. bodyweight 1200-1800g,showthe samelobulationandramifica- tionofthe conductive bronchialtree. Allfourrodentspossess dorsallyopencartilaginousbracesinthe trachea and in both main bronchi up to the first ventral branch of the left lung or to the emerging bronchus ofthe right middle lobe. The distribution ofcartilage tissue inthe bronchialtree ofthe four speciesinvestigatedisidenticalandshowsnorelationtothe size ofthelungorthebronchi.The defini- tionoftheterm"bronchus"bythepresenceofcartilageiscriticizedandthefunctionofintrapulmonary bronchialelementsisdiscussed. Key words: Rodentia, lung, airways, bronchi, cartilage Introduction The generally assumed function of cartilage in any airway System is to keep the airways open and to prevent their collapse. In the literature the subject of the function of air- way cartilage has been completely ignored. The present investigation determines the dis- tribution of bronchial cartilage in the conductive bronchial tree of mammals of close phylogenetical relation, similar body proportions, comparable locomotory habits. and identical lung anatomy, differing mainly in their adult body size (Gehr et al. 1981). Pre- vious studies of the author proved the four considered species to yield a good basis for this comparison. The lungs of Micromys, Mus. Rauns, and Cricetomys share all basic morphological parameters. The lung volumes of all four species are isometrical to their body weight. Micromys, Mus, Rattus, and Cricetomys all show an identical pattern of bronchial ramification and of lung lobulation (Valerius 1996). The influence of body size on the cartilaginous stiffening of the bronchi was expected to enlighten the func- tional relations under which these chondroid elements have evoked. In compensation for the term "ring'* the term "brace" or "C-shaped brace" is used in the present study for the larger extrapulmonary cartilaginous elements. According to the generally used nomenclature, smaller. irregulär chondroid elements are called ""plates" (Vanpeperstraete 1973). Distributionofbronchialcartilageinmyomorphrodents 221 Material and methods Frompreviousstudies(Valerius 1996;Dieterlen 1988),thebiologicaldatafortheharvestmouse,Mi- cromysminutusPallas, 1771,thehousemouse,MusmusculusLinnaeus, 1758,thebrownrat,Rattusnor- vegicus Berkenhout, 1769, and the giant pouched rat, Cricetomys gambianus Waterhouse, 1840, are listedintable1. The givenmeanvalueswere takenfromadult animalsofbothsexes. Foradescription ofthemethodforpreparingtheSiliconrubbercasts,seeValerius(1996). Table1. Meanbodyweights, totalbodylengths andnose-rump lengths ofMicromys, Mus, Rattus, and Cricetomys. Species(n) bodyweight totalbodylength nose-rump-length Micromys(15) 6.6g 111mm 56mm Mus(15) 35.9g 198mm 102mm mm mm Rattus(14) 255.9g 394 211 mm mm Cricetomys(13) 1447.5g 693 357 Micromys is one ofthe smallest mammals, whereas Cricetomysis a giant myomorphrodent and a medium-sized mammal. The lung sizes ofallfourspecies are comparedinfigure 1, the ramification of the bronchiinthe left lung as representedbybronchialcasts is shown infigure2. Micromys,Mus, and Rattus are genera of the family Muridae, while the taxonomy of Cricetomys is subject to discussion. ThisanimalmaybeplacedeitherinthefamilyofCricetidaeorinaseparatefamily,the Cricetomyidae (Dieterlen1988).Itslunganatomyisinallregardssimilartothatoftheotherthreespecies. Fig.1. Freeze-dried lungs of Micromys (A), Mus Fig.2. Silicone rubber cast of the airways of the (B), Rattus (C), and Cricetomys (D), showing the left lungs of Micromys (A), Mus (B), Rattus (C), identical lobulation and the size relations of the and Cricetomys (D), view from ventral. Scale bar lungs in the ventral view. Scale bar represents represents10mm. 100mm. 222 K.-P.Valerius Forthe purpose ofthis studythree individualseach of Micromys, Mus, and Cricetomys and four Rattus weresacrificed,thelungswerestainedandcomparedto lungcastsofthesamespecies. All animals were killed by exposure to C02 and placed in a supine position. The trachea was opened and a cannula inserted. According to a method after SimsonandvanHorn (M. Güntert,Bern,Switzerland, pers.comm.)thewholeembryoswererenderedtranslu- cent. The lungs were excised from the thorax and filled withaSolutioncontaining80volume% 96% alcohol,20 volume% glacialaceticacidand30mgAlcianblue.The lungs were fixed and stained for two days, being filled with and submerged in this Solution. Then, the speci- mens were dehydrated in 99% alcoholforthree to five Fig.3. Schematic drawing explaining the days andmaceratedin a 1% KOHSolutionfor another method of counting of the cartilage braces few days (until the lung becomes soft and lucent). In ofthetrachea.Inthisventralviewthere are thelaststep,thespecimenswereimmersedinaSolution three free endings of braces on the right of 79 vol.% bi-distilled water, 20 vol.% glycerine and side and four free endings on the left side 1% KOH until they were completely translucent. The leadingto an average of3.5 bracesinthe il- specimens could be stored in a Solution of 50 vol.% lustratedpartofthetrachea. 96% alcoholand50% glycerinforlongperiods. The translucentspecimenswere then analysedwith the help of a Stereo microscope and the dorsal endings ofthe cartilaginousbraceswere counted on the right and the left side and the average was calculated (Fig.3).Further,thecartilaginouselementsintherightandintheleftlungwerecounted. Thelengthofthetracheawasmeasuredfromthelowermarginofthecricoidtothebifurcationinto the mainbronchi. The outer diameter ofthe trachea was taken in the middle ofthe distance between thecricoidandthebifurcation. Results In all species included in this study, only dorsally opened cartilaginous braces were found in the trachea and the main bronchi. Closed rings of cartilage encircling a bronchus could not be detected in any specimen. All four rodents showed the same distribution of chon- droid braces over the identical parts of their conductive bronchial tree, regardless of the size of the lungs or the diameter of the bronchi. Figure 4 shows the stained translucent lungs ofMicromys, Mus, Rattus and Cricetomys, all enlarged to the same size. For the ori- ginal size relations ofthe lungs compare with figures 1 and 2. Cartilage in the tracheal wall These four rodent species possess dorsally open cartilaginous C-shaped braces in the tra- chea. The number of these chondroid elements differed among the species. Micromys and Mus showed 14 and 13 braces, respectively, the larger species Rattus and Cricetomys both had an average of 24 elements (Tab. 2). The bifurcation of the trachea into the two main bronchi was not supported by cartilage in the carina. Cartilage inthe bronchial walls Both main bronchi possess cartilage up to the site of the first bifurcation. In the left lung, the cartilaginous braces of the main bronchus extend down to the point where the first ventral branch leaves the main bronchus, and 4-5 small cartilaginous elements can Distributionofbronchialcartilageinmyomorphrodents 223 Fig.4. LungsofMicromys (A),Mus (B), Rattus (C), and Cricetomys (D). Translucent specimenshow- ingthedistributionandformofairwaycartilage.Allspecimensarebroughttoidenticalsize.Eachscale barrepresents10mm. be found in this first ventral branch. In the right lung, the braces join the main bronchus down to the emerging bronchus of the middle lobe. Again, 3-4 cartilaginous elements can be found on this middle lobe bronchus (Tab. 3). In all specimens deserved, no cartilages could be detected in the carina. In the case of a bifurcation, the chondroid elements were placed in the wall opposite to the leaving branch, facing the carina (Fig. 5). Figure4 shows the distribution of bronchial cartilage in the respiratory tracts of the four species. 224 K.-P.Valerius Table2. Diameterandlengthoftrachea,inabsolutetermsandin % ofnose-rumplength,andnumber ofcartilagineousbracesinthetracheaofMicromys,Mus,Rattus,andCricetomys. Species(n) outerdiameterof % ofnose-rump lengthoftrachea % ofnose-rump- trachea length length Micromys(3) 2.0mm 3.6% 6.8mm 12.2% Mus(3) 2.0mm 2.0% 13.0mm 12.7% mm mm Rattus(4) 4.1 1.9% 30.8 14.6% mm mm Cricetomys(3) 6.3 1.8% 45.7 12.8% Table3. Number of tracheal and bronchial cartilage braces in Micromys,Mus,Rattus,andCricetomys. Species(n) bracesinthe bracesin bracesof leftmain rightmain trachea* bronchus bronchus Micromys(4) 6.8 5.5 14.0 Mus(3) 8.0 5.0 13.7 Rattus(4) 8.5 9.5 24.0 Cricetomys(3) 8.4 6.0 24.0 meanofleftandrightbraceendingspertrachea Fig.5. Schematic drawing explain- Discussion ing the distribution ofcartilage ele- ments in a lobar bronchus exiting Functionalaspects from the main bronchus. The arrow indicates the position ofthe carina. The diameter of the airways and the stiffening of the See alsoFig.4. airway walls by cartilaginous braces do not correspond, at least not when the trachea and the extrapulmonary bronchi are compared to the intrapulmonary bronchi, and when the intrapulmonary bronchi are compared to each other according to their positions and diameters. All extrapulmonary airways in all four species are equiped with cartilage, regardless of their diameter. All intrapulmonary airways lack cartilage, at least, they possess no chondroid tissue soon after the bronchus enters the lung tissue. Vanpeperstraete (1973) described principal differences in the form ofintrapulmonary and extrapulmonary airway cartilage for several mammalian species (rat, dog, sheep, cow, pig, horse). He found regulär braces in the trachea and main bronchi and described a sharp boundary to the irregulär cartilage elements surrounding the intrapulmonary bronchi. Extrapulmonary airways have regularly arranged, open C-shaped braces of cartilage. The dorsal part ofthe airways always remains free ofcartilage, so that the airway is never completely surrounded by skeletal elements. The smooth airway musculature connects the open ends of the braces. For this reason, cartilage and muscles are arranged in the same layer ofthe airway wall. Extrapulmonary airways in all mammals require cartilage stiffening in order to re- main open under differing ambient pressures. The consequences of a pathological destabi- lisation of the tracheal cartilage elements in man reflects this function (Riede and Costa- A bel 1993). softening of the tracheal cartilage braces, tracheomalacia, results in a compression ofthe trachea known as "scabbard trachea" with a narrowing ofits lumen. Distributionofbronchialcartilageinmyomorphrodents 225 Intrapulmonary airways in contrast show irregulär elements, called plates. A Single element is never closed around a bronchus in land-living mammals, but the plates cover the entire circumference of the bronchi. The musclature of these intrapulmonary bronchi in man lies between the epithelium and the cartilage as a closed layer, so that muscles and cartilage form two clearly separate layers of the bronchial wall (Duncker 1994). The cartilage elements in the intrapulmonary bronchial tree do not supply sites of fixation to the bronchial musculature. These findings point to different functional parameters in extrapulmonary and intra- pulmonary airway wall structure. The intrapulmonary bronchial skeleton consisting ofconnective tissue and irregulär car- tilaginous plates is arranged external to the bronchialmusculature, the latter determinig the diameter ofthe bronchi. These plates are notsuitable formaintaining apatent bronchusbe- cause theydonotregularlyembracemorethanhalfofthe circumference ofthe bronchi. The mechanism for maintaining intrapulmonary bronchi in an open condition in land- living mammals is the traction from the expanded lungs acting on these bronchi and the elastic retraction force of the respiratory lung tissue itself. The bronchial tree of mam- mals, apart from the function of distributing the oxygenated air as evenly as possible over the exchange surface, fulfills a second function, i.e., it is the main skeletal structure ofthe soft flexible lung tissue. It is hypothesized here, that when intrapulmonary airways in land-living mammals possess cartilage, as for example in man, it helps to prevent overex- pansion ofthe bronchi and, additionally, acts as a fixation for the connective tissue. which stabilizes the inner structure ofthe lungs. As a consequence ofthis function, an instability ofthe intrapulmonary bronchial wall by distruction or aplasia of the cartilage plates leads to a widening ofthe bronchi, the condition known as bronchiectasis. Nomenclatureaspects In the human anatomical nomenclature (Warwick and Brookes 1989), the terms "bronchus" and "bronchiolus" are defined by the presence or absence of cartilage, re- spectively, in the airway walls (Duncker 1994). According to this terminology obviously homologous parts of the bronchial tree of different animals have to be called "bronchi" in man and other larger mammals, but "bronchioli" in smaller species, simply because of the presence or absence of cartilage. For interspecific comparative investigations and de- scriptions of the mammalian lungs, a distinction between bronchi and bronchioli cannot be based on the criterion of cartilage in the wall. For example, in the myomorph rodents studied here, the principal airway of each lung should be called the main bronchiolus. The use of the diminutive for the largest intrapulmonary airways of an animal does not appear appropriate. On the other hand, in marine mammals the chondroid rigidity of the airways even includes the terminal bronchioles. In these marine forms cartilaginous spir- als or closed rings occur (Belanger 1940; Denison et al. 1971; Engel 1956; Fiebiger 1915/16; Kooyman and Sinnett 1979; Wislocki 1929, 1942). They obviously keep the air- ways open against external water pressure. The distinction between bronchi and bronchioli should be limited to human anatomy, where the term "bronchioli" has a long tradition. Neither the distribution ofcartilage, nor the diameter, the epithelial lining, or the types of glands present, or any other morpholo- gical structure yields a basis for the definition of the term "bronchioli" that would be ap- plicable to mammals of different body size or adapted to different environments. In con- sequence, the last generations of airway branches before opening into the acini should be called terminal bronchi and respiratory bronchi. The term "cartilaginous ring" should be avoided as long as the element does not form a closed ring. To my knowledge, no such closed rings have ever been described in any tra- chea or bronchial tree ofland-living mammals. 226 K.-P.Valerius Acknowledgements Thanks to Prof. Dr. Dr. H.-R.Duncker (Giessen) for his support ofthis work in all respects, to Prof. Dr. P.Langer (Giessen) forhiscontinuedencouragement, andtoDr. R.Snipes (Giessen)forlinguistic advice. The quality of the figures will convince the reader of the excellent technical assistance of Ms. M. Gottwald(Giessen). Zusammenfassung Größenunabhängige VerteilungvonBronchialknorpelnbeivierArtenmyomorpherNagetiere Die Verteilung und die Form knorpeliger Stützelemente in der Wand der Luftwege von vier verschieden großen mäuseartigen Nagetieren wurden an Hand von knorpelgefärbten Aufhellungs- präparaten dargestellt. Die Trachea und die extrapulmonalen Bronchen der euroasiatischen Zwerg- maus, Micromys minutus, der Hausmaus, Mus musculus, der Wanderratte, Rattus norvegicus und der Gambia-Riesenhamsterratte, Cricetomys gambianus, weisen ausschließlich dorsal offene, regelmäßig angeordnete Knorpelspangen auf. Innerhalb des rechten Lungenflügels dehnt sich die Knorpelaus- steifung mit wenigen Elementen bis auf den Abgang des Mittellappenbronchus vom Hauptbronchus aus, auf der linken Seite bis auf den ersten großen ventralen Bronchus, der den linken Haupt- bronchus verläßt. Bei allen vier Arten setzt sich die Knorpelauskleidung bis zu einem identischen Punkt im Verzweigungsgefüge des Bronchialbaums fort und nicht bis zu einem bestimmten Durch- messer eines Bronchus. Aufgrund der sehr uneinheitlichen Knorpelverteilung in den Lungen der Säugetiere verschiedener Familien, Körpergrößen und Lebensweisen sollte der Begriff „Bronchio- lus" in der zoologischen und veterinärmedizinischen Terminologie nicht gebraucht werden. Es gibt keine Kriterien, die eine artübergreifend sinnvolle Definition des Terminus „Bronchiolus" ermögli- chen könnten. Nach der Funktion der Knorpelelemente in der Bronchialwand landlebender Säuger sollte unterschieden werden zwischen extrapulmonalen Knorpelspangen, die das Lumen der Luft- wege offenhalten, und intrapulmonalen Knorpelelementen, die zur Stabilisierung der Bronchen ge- gen den sie weitenden Zug und als Ansatz für das die innere Lungenstruktur stabilisierende Binde- gewebe dienen. 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