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The structure, composition and elastic properties of the teleost bulbus arteriosus in the carp, Cyprinus carpio
Comp. B&hem.
Physiol., 1973, Vol. 46A, pp. 699 to 708. Pwgamon Press. Printed in Great Britain
THE STRUCTURE, COMPOSITION AND ELASTIC PROPERTIES OF THE TELEOST BULBUS ARTERIOSUS IN THE CARP, CYPRINUS CARPIO*
J. HAMILTON
LICHT
and WILLARD
S. HARRIS
Abraham Lincoln School of Medicine, University of Illinois at the Medical Center, Department of Medicine, Section of Cardiology, Chicago, Illinois 60612, U.S.A. (Received 5 January 1973) Abstract-l. Pressure-volume studies show that the bulbus arteriosus of carp (Cyprinus c&o) is extremely distensible and resilient. 2. Studies with specific elastic tissue stains, fluorescence and elastase show that the bulbus has abundant elastic tissue. 3. Bulbar elastic tissue differs from that of mammalian aorta in its coloration by non-specific stains, lack of lamellar distribution and marked solubility in hot 0.1 N NaOH and warm 89% formic acid. 4. Smooth muscle cells in the bulbus probably synthesize its elastic tissue.
INTRODUCTION THE BULBUS ARTERIOSUS
of the teleost is a thick-walled chamber that connects the single cardiac ventricle to the ventral aorta. Because of its central location in the single circulation of the fish, the bulbus can regulate the impedance faced by the ejecting ventricle and the pressure and flow occurring in the systemic arteries and in the gill capillaries. During systole the teleost ventricle ejects its contents into the bulbus arteriosus, which distends. During diastole the ventriculobulbar valve is closed, but forward flow into the ventral aorta continues as the bulbus contracts down to its previous size (Mott, 1950). The bulbar maintenance of forward flow during ventricular diastole is important in teleosts because of their long diastolic periods. The bulbus also attenuates peak systolic intraventricular pressure, thereby protecting the delicate gill capillaries from excessive distention and possible rupture. Because it buffers the otherwise wide swings of systolic and diastolic aortic pressure, the bulbus has been described as a “windkessel” (von SkrarnIik, 1935) or “pressure chamber” (Johansen & Martin, 1965). Despite the prominence and well-recognized importance of the bulbus in the teleost circulation (Johansen, 1962; Satchell, 1971), the structure, composition and elastic properties of the bulbar wall have not been clearly defined. l This investigation was supported in part by American Heart Association grants, U.S. Public Health Service Research Grants Nos. HE14412-02 and lT12-HL05879 and a U.S. Public Health Service general research support grant.
699
700
J. HAMILTON LICHT AND WILLARD S. HARRIS
Strictly, in physical terms, elasticity means resistance to deformation. More commonly, and in biology, elasticity denotes a combination of stretchability, i.e. low resistance to deformation, and complete reversibility of deformation upon removal of the deforming force, or stress (Burton, 1954; Landowne & Stacy, 1957). In this common usage, the bulbus is highly elastic. The elasticity of the bulbus has not previously been quantitated, its structure and histological staining characteristics have received but brief mention (Parsons, 1930; Abraham, 1969), and only Lansing (1959), in a terse comment on the bulbus of the anglerfish, Lophius piscatorius, has discussed the similarity and dissimilarity of elastic tissue of the bulbus to that of mammals. We have, therefore, investigated the stress-strain properties of the bulbar wall by pressure-volume studies, documented its histological and tinctorial characteristics using several different stains, determined the effects produced by elastase on two known characteristics of elastic tissue-its fluorescence and affinity for orcein (Ayer, 1964) and quantitated the amount of elastic tissue extractable from the bulbus by conventional extraction methods (Lowry et al., 1941; Hass, 1942). Because the carp (Cyprinus carpio) is an easily accessible and common fresh-water teleost, its bulbus arteriosus was studied. When appropriate, comparison studies of carp ventral aorta, human and canine proximal aortae, and elastic tissue from bovine ligamenturn nuchae were also done.
MATERIALS
AND
METHODS
Carp, Cyprinus curpio, from the upper Mississippi River were kept for several months in a continuously aerated, 6000-gal holding tank at lo-15°C and were fed chopped smelt and horse heart strips twice weekly. For about 2 weeks before study they were kept in our laboratory in a continuously filtered and aerated tank, which was equipped with a cooling unit (Living Stream Model LS-700, Frigid Units, Inc., Toledo, Ohio) that maintained water temperature at 20°C. Human proximal aortae were obtained at the post-mortem examination of three women, 39, 58 and 63 years old. Proximal aortae were excised, immediately after death, from pentobarbital-anesthetized dogs killed by transthoracic intracardiac injection of 100 cm3 air. Elastic tissue fragments from bovine ligamentum nuchae (Schwarz/Mann, Orangeburg, N.Y.) and swine elastase (65 units/mg, code ESFF, Worthington Biochemical Corp., Freehold, N.J.) were purchased. One unit of elastase solubilizes 1 mg elastin in 20 min under specified assay conditions (Sachar et al., 1955). Statistical analysis was done with Student’s t-test (Snedecor, 1956).
Pressure-volume
studies
Six carp, weighing 0.8-l .2 kg, were killed by a crushing blow to the head. Through an incised triangular flap of the ventral wall cephalad to the pectoral fins, the pericardium was cut and the ventricle, bulbus and ventral aorta excised as a unit. Bisection of the ventricle perpendicular to its long axis exposed the ventriculobulbar orifice. Through this orifice, the bulbar and ventral aortic lumina were flushed by syringe three times with 5 ml of carp Ringer solution, which contained (in mM/l.) Caa+, 2.3 ; Naf, 156; Kf, 3.1; and Cl-, 160. The solution was buffered at pH 7.5 with NaH,PO, and Na,HPO, and, as determined with a freezing-point-depression osmometer (Advanced Instruments Co., Newton Highlands, Mass.), had an osmolality of 301 mOsmoles/l.
STUDY
OF THE
TELEOST
BULBUS
ARTEXIOSUS
IN
THE
CARP
701
The ventriculobulbar orifice was closed by a silk ligature tied tightly around the ventriculobulbar groove. The blunt tip of a lo-gauge, stainless steel needle was passed retrograde through the ventral aorta into the bulbar lumen and secured in place with a silk ligature. The needle hub was attached to a stainless steel Y-adaptor. A 5-ml syringe, connected by a three-way stopcock to a second arm of the adaptor, was used for intrabulbar injections and withdrawals. The tip of a saline-filled polyethylene catheter (length 8 cm, id. O-58 mm) was passed through the third arm of the adaptor and the needle and into the bulbar lumen. The catheter hub, which closed off the orifice of the Y-adaptor, was attached directly to a Statham P23Db strain gauge transducer. The transducer, lixed horizontally level with the catheter tip, was connected to the SGM-2 pressure amplifier channel of a photographic recorder (Electronics for Medicine, White Plains, N.Y.). Full-scale pressure on the recording was 100 mm Hg and paper speed was 5 mm/set. The bulbus was suspended in a bath of oxygenated carp Ringer solution at 22°C. Before study air was evacuated from the bulbar lumen, needle and Y-adaptor by withdrawing the plunger of the empty syringe attached to the three-way stopcock until resistance permitted no further withdrawal. The stopcock was closed toward the Y-adaptor, sealing the evacuated chamber. The syringe was replaced with a 5-ml syringe filled with carp Ringer solution, which was injected until intrabulbar pressure was 0 mm Hg relative to the atmosphere. A trial consisted of adding to the intrabulbar volume at 0 mm Hg, which measured approximately 0.1 ml, five consecutive injections of 0.4 ml oxygenated carp Ringer solution, for a cumulative injectate of 2 ml, followed by five consecutive withdrawals of 0.4 ml solution each. Each step increment or decrement of volume was made in 1 set, and the bulbus was kept at the new volume for another 3-4 set before the next step change was begun. Each of the six bulbi received six to twelve trials for a total of fifty-one trials. Histologic studies Bulbi arteriosi and ventral aortae, freshly excised from two carp, and one human proximal aorta were fixed for 12 hr in 10% neutral formalin and embedded in paraffin. Nuchal elastic tissue fragments were parafhn-embedded both with and without a previous 12 hr formalin fixation. Paraffin sections, 6 /J thick, from each of the five kinds of tissue preparations were simultaneously stained in the same tray. Stains used were hematoxylin and eosin, Gomori and Masson trichrome for connective tissue, Bielschowsky silver impregnation for reticulin and nerve fibers, alcian blue for acid mucopolysaccharides and seven elastic tissue stains: Fraenkel orcein, Taenzer-Unna orcein, Weigert resorcein fuchsin, Verhoeff iron hematoxylin, Gomori aldehyde fuchsin, Gallego iron fuchsin and Mallory phosphotungstic acid hematoxylin (PTAH). Staining methods were done as described by the Armed Forces Institute of Pathology Manual of Histologic and Special Staining Technics (1949) except for the two orcein stains (Lillie, 1965) and the silver impregnation (Nassar & Shanklin, 1961; Hirsch et al., 1970). The effects of elastase on staining with orcein and on fluorescence were assessed in frozen sections, 12 p thick, of carp bulbus arteriosus and ventral aorta and canine proximal aorta placed on glass slides. One set of sections was immediately fixed in 10% formalin. The second set was layered over by pipette with 0.1 mg elastase, dissolved in 0.1 ml of 0.2 M Tris buffer at pH 8.8 (Sachar et al., 1955), and was placed in 50-ml plastic syringes, which were laid flat in an oven. Evaporation of the elastase solution was inhibited by the addition of 5-10 ml water to the bottom of the syringes, which were then sealed. After 30-min incubation at 37”C, the sections were gently rinsed with distilled water to remove enzyme and solubilized protein and fixed in formalin. Both the untreated and elastase-treated sets were stained by the Fraenkel orcein method. Other sections from both sets were mounted, unstained, between a glass slide and cover-slip with a polyvinyl alcoholbuffered saline-glycerol medium (Rodriguez & Deinhardt, 1960), and were examined for fluorescence with a Zeiss fluorescence microscope equipped with an immersion condenser
702
J. HAMILTON LICHT AND WILLARD S. HARRIS
(l-2/1.4 N.A.). When nuchal elastic tissue fragments were examined with two BG12 exciter filters (spectrum of transmission 320-510 rnp) present, the fragments fluoresced a brilliant blue-white. However, when this fluorescence was passed through a barrier filter that excluded frequencies below 470 rnp, nuchal elastic tissue fragments fluoresced a yellow-green. In the present study this yellow-green fluorescence was used to identify elastic tissue.
Extraction studies Tissues were minced with scissors and homogenized in S-10 ml of 95% ethanol in a The paste was transferred to 50 ml Pyrex Waring blendor until they were paste-like. centrifuge-tubes, refluxed for 30 min over steam in 40 ml of 95% ethanol and centrifuged at 1750 g, and the supernatant was decanted. Ethanol refluxing, centrifugation and decantation were repeated. After an ether rinse and oven-drying at 95°C for 24 hr, samples of the defatted-dried tissues were weighed. Defatted-dried bulbus arteriosus, ventral aorta and cardiac ventricle from carp, human proximal aorta and canine proximal aorta were treated by the quantitative elastin extraction method of Lowry et al. (1941), which is based on the insolubility of elastic tissue and the solubility of other tissue constituents in hot alkaline solution. Tissue samples, weighing 0.18-1.90 g, were mixed with 40 ml of 0.1 N NaOH in clean 50 ml Pyrex centrifuge-tubes, heated for 10 min at 95°C with intermittent stirring in a boiling water-bath, centrifuged for 5 min at 1750g and decanted. The precipitate was again heated for 10 min in 40 ml of 0.1 N NaOH and the mixture was centrifuged and decanted. After three centrifugerinses-each consisting of a rinse with distilled water, centrifugation for 5 min at 1750g and decantation-the precipitate was oven-dried for 24 hr at 95”C, and the resulting residue was weighed. The residues from the bulbus and human proximal aorta were examined for fluorescence. The percentage of defatted-dried tissue weight that is residue was determined as 100 times the weight of residue/weight of defatted-dried tissue. The percentage of residue weight due to inorganic constituents was determined either by incubation in dilute (0.1 N) formic acid for 8 hr at 22°C which dissolves inorganic elements, such as calcium and phosphorus (Dempsey & Lansing, 1954), or by ashing over an open flame in predried porcelain crucibles. Either the weight lost with 0.1 N formic acid or the weight of the ash was taken as the weight of the inorganic constituents. Hass (1942) reported that elastic tissue was quantitatively extracted from other tissue constituents with warm concentrated (89%) formic acid. Defatted-dried carp bulbus arteriosus, carp ventral aorta and human proximal aorta were incubated for 72 hr in 890,‘, formic acid (1 ml/5 mg tissue) at 45” C in sealed Pyrex flasks. After repeated centrifugerinses with distilled water in clean Pyrex centrifuge-tubes, the tissues were dried for 24 hr at 95” C and the resulting residues were weighed and examined with the fluorescence microscope. RESULTS
Pressure-volume
studies (Fig. 1)
After each step increase of volume, intrabulbar pressure quickly declined from the immediate postinjection level. During the first second postinjection, the pressure fell on average approximately 0.5 mm Hg and remained relatively stable over the next 2-3 sec. The intrabulbar pressures used for the graph in Fig. 1 were measured 3 set after the step change in volume. Data for each bulbus were taken as the mean of all its trials. These means are averaged, with sample N = 6, in the curve in Fig. 1, which relates pressure to the changes in intrabulbar volume for the group. With either increments or
STWXW OF THE TELEOST IBULBUS ARTERXQSUS IN THE CARP N=6
FIG,
1.
7a3
CARP
The re&t~on of intrabutbatr pressure to c-s in intrabdbar volume. Iatrabulbar volume at 0 mm Hg averaged O-1 ml.
decrementsof v~~~rne~ the curve is steepestbetween 0 and O-4ml changes in vohmie. Thereafter, the curve is more gradua& with pressure changing an average -t_S.E, of 4.8 + 0-4s mm Hg per O-4 ml change oE volume. Hysteresis is present but is not significant at the 5 per cent level at any change in volume.
The w& of the n~n~tended bulbus has three distinct coats {Fig. 2)_ The outer layer, or adventitia, which is very thin, is composed of loose fibrous connective tissue, primarily colIagen, and contains medium-sized arterioles, venules and lymphatic vessels, occasional smooth muscle fibers, rare small. nerve bundles, and smaII amounts of fat. The inxrer coat, or intima, is in some areas limited to an cndothelial lining of cuboid or Bat cells but, in other areas is a thin spongy layer, which is composed p~ar~~y of connect&e tissueS is rich in nerve fibers and contains many small capillary&ke channels. The tunica media, occupying more than 90 per cent of the unstretched bulbar wall, is much thicker in proportion to the vessel wall than is the media of mammalian elastic arteries. There is much coaaective tissue throughout the bulbar media, which also contains a circumferential& arranged layer of ~rn~~ct smooth muscler and a thicker, Ioosefy arranged internal layer of ~terdig~~~ smooth rnzfscfe far&&s. Finger-auks projections of adventitia carry vasa vasorum to the compact external smooth muscle layer of the media. In addition to having a small amount of collagen, colored blue with Masson trichromq the bulbar media is extraordinarily rich in tissue that stains strongly with or&n (Fig, 3)* Weigert resorcein fuchsin, VerhoeE iran hernato~~~~ Gallego iron fuchsin and Gomori aldehyde fuchsin, which are stains with some
704
J. H.&MILTON LICHT
AND
WILLARD S. HARRIS
specificity for elastic tissue (Dempsey & Lansing, 19.54; Pearse, 1968). These stains impart the same color to the elastic tissue, whether in the media of the carp bulbus, ventral aorta, human proximal aorta or bovine ligamenturn nuchae. Each elastic tissue stain colors the bulbus much more deeply and extensively than it does the media of the human proximal aorta or the carp ventral aorta. In contrast, the bulbar elastic tissue differs from that of mammalian specimens in its coloration by other stains. The bulk of elastic tissue in the carp bulbus and ventral aorta stains light green with Masson trichrome and does not stain with PTAH or eosin. The bovine nuchal and human aortic elastic tissues stain red with Masson trichrome, deep blue with PTAH and pink with hematoxylin and eosin. The bulbar media does contain a few fine eosinophilic fibers and a few 0 verall, however, the bulbar media is fine fibers that stain blue with PTAH. richly basophilic with hematoxylin and eosin, while the media of the human Owing to its content of acid mucopolysaccharides proximal aorta is eosinophilic. (Gore & Larkey, 1960), the media of human proximal aorta, except for its elastic laminae, stains richly blue with alcian blue. In contrast, the carp bulbs and ventral aortic media stain only slightly blue with alcian blue. Bielschowsky silver impregnation, with and without counterstaining by the shows that reticulin and nerves are elastic tissue stain, Gallego iron fuchsin, present, particularly in the intimal and subadventitial regions of the bulbus, and that these argyrophilic elements are clearly different from the bulbar elastic tissue. The formalin-fixed fragments of nuchal elastic tissue had the same staining characteristics as the nonfixed nuchal fragments. When frozen sections were examined with the fluorescence microscope, elastic tissue of the carp bulbus and ventral aorta, like the nuchal elastic tissue and the elastic laminae of the canine proximal aorta, fluoresced vividly yellow-green. Application of elastase to the frozen sections of all three tissues reduced the widespread, deep yellow-green fluorescence to a small amount of yellow granular debris (Fig. 4) and abolished the staining response to orcein. Medial elastic tissue in the bulbus appears to differ morphologically from that In man and dog, the elastic tissue is in the human and canine proximal aortae. concentrated in laminae, from which fine elastic tissue fibers emanate. The media
FIG. 3A. Part of the internal layer of bulbar media stained for elastic tissue, which appears black here. The light-grey region (lower left) is collagen-rich ventriculobulbar valvular tissue, included here for contrast. (a) Internal layer of bulbar media. Taenzer-Unna orcein stain (X 112). B. (b) Bulbar lumen. (c) Valvular tissue. Note the dark staining elastic laminae. Elastic tissue in human proximal aorta. (a) Media. (b) Intima. Taenzer-Unna orcein ( x 112). The carp bulbus in (A) and the human proximal aorta in (B) were stained simultaneously in the same tray and were photographed and developed under identical conditions. The bulbus stains more intensely and extensively with specific elastic tissue stains than does the human aorta. C. Higher magnification of the valvular region shown in (A). The arrows at (a) point to the amorphous elastic tissue. (b) Coarse elastic fibers. The arrows at (c) point to fine elastic fibers. ( x 440.)
FIG. 2A. Part of a cross-section of the nondistended bulbar wall with the pericardium removed. (a) Bulbar adventitia. (b) External layer of bulbar media containing compact smooth muscle. (c) Internal layer of bulbar media containing more loosely arranged smooth muscle. (d) Bulbar intima. (e) Bulbar lumen. (f) Blood in the bulbar lumen. (g) Ventriculobulbar groove. (h) Ventricular myocardium. Gomori trichrome ( x 20.5). B. Whorls of smooth muscle in the internal layer of the bulbar media. This stain does not show the rich elastic component of the bulbar wall. Gomori trichrome. ( x 262.)
FIG.
3
(a)
(b)
FIG. 4. Effects of elastase on fluorescence of elastic tissue in frozen sections. Fluorescence appears white here. The top three panels show untreated elastic tissue, while the bottom three panels show the same tissues after 30 min incubation with elastase. (a) Canine proximal aorta. Note the thick elastic laminae. (b) Carp ventral aorta with laminae. (c) Carp bulbus arteriosus. For clarity, a trabeculated area of bulbar media near the lumen is shown; the black lacunae are areas of the lumen. In all three tissues, as the lower panels show, elastase treatment reduces the fluorescence to a small amount of granular debris. (Approximately x 115.)
705
STUDYOF THBTBLBOSTBULBUSABTBRIOSUS IN THE CARP
of the carp bulbus, by contrast, is pervaded throughout by both amorphous tissue and numerous coarse and fine fibers, all of which stain deeply with the elastic tissue stains. Each smooth muscle cell of the bulbus appears to be enveloped by a mantle of elastic tissue. Unlike the bulbus, the carp ventral aorta contains elastic tissue laminae, which resemble closely those found in the human and canine proximal aortae. To a distinctly lesser extent than in the bulbus, the carp ventral aortic media is also pervaded by nonlamellar elastic tissue. Extraction
studies
As Table 1 shows, the percentage of defatted-dried tissue weight remaining as residue after 0.1 N NaOH extraction was (mean+ S.E.) 0.8 f 0.12 per cent for carp bulbus arteriosus, 8.9 _+2.31 per cent for carp ventral aorta, 39.6 + 1.45 per cent for human proximal aorta and 32.6 per cent for canine proximal aorta. Defatted-dried carp ventricular tissue (1.90 g) had no measurable elastic tissue residue. Inorganic constituents, determined by O-1 N formic acid treatment or by ashing, averaged 47.4 per cent of carp bulbar residue, 9.8 per cent of the carp ventral aortic residue and 12.1 per cent of human proximal aortic residue. Therefore, bulbar, carp ventral aortic and human proximal aortic residue weights were multiplied by O-526, 0.902 and O-879 respectively to obtain weights corrected for TABLE ~-WEIGHT OF RESIDUEMTBACTBDWITH 0.1 N NaOH OR 89% FORMICACID
Tissue
No. of determinations
Total number of specimens
Residue weight * (%)
Residue after correction for inorganic content* (%)
O-1 N NaOH Carp bulbus arteriosus Carp ventral aorta Carp cardiac ventricle Human proximal aorta Canine proximal aorta
105 29 7 3 1
10 3 1 3 1
O-8 * 0.12f 8.9 + 2.31 0.0 39.6 f l-45 32-6
0.4 !I 0.06 8.0 f 2.07 34.8 + 1.27
89% formic acid Carp bulbus arteriosus Carp ventral aorta Human proximal aorta
7 2 3
* Percentage residue weight =
109 15 3
9.3 f 1.52 18.4 44.1 iz 4.56
weight of dried residue (mg) weight of defatted-dried tissue (mg)
x 100.
t Percentage residue weight after correction for inorganic content = per cent residue weight x the appropriate correction factor, which is given in the text. $ Mean + S.E. with the number of determinations used as the sample iV.
706
J. HAMILTON
LICHT AND WILLARD S. HARRIS
inorganic content. Corrected values, expressed as percentage residue after correction for inorganic content, are given in Table 1. Residues from bulbus and human proximal aorta, which were the only ones examined by fluorescence microscopy, fluoresced vividly yellow-green. With human proximal and carp ventral aortae, extraction with 89 per cent formic acid proceeded as described by Hass (1942). However, during centrifugerinsing of the bulbar tissue, approximately 15 ml of a translucent gel, or coagulum, which entrapped fine tissue fragments and fibers, formed and persisted despite repeated rinsing. For elimination of the gel, it was oven-dried for 24 hr at 95°C rehydrated with 40 ml of distilled water and heated for 30 min at 95°C in a boiling water-bath. After centrifugation and redrying, the samples were reweighed. The bulbar residue was 9.3 _t 1.52 per cent of the defatted-dried tissue weight (Table 1). The carp ventral aortic and human proximal aortic residues were 18.4 per cent and 44.1 k 4.56 per cent of defatted-dried tissue weight, respectively. The residues from each of the three kinds of tissues fluoresced vividly yellow-green.
DISCUSSION
In human thoracic aorta, the volume at 7.3 mm Hg pressure, which was 9 ml, was found to increase 22 per cent when pressure was raised to 33 mm Hg (Hallock & Benson, 1937). In the present study (Fig. l), intrabulbar volume (0.3 ml) at 7.3 mm Hg was found to increase 700 per cent when pressure rose to 33 mm Hg. In this range of pressures, therefore, the carp bulbus arteriosus is about thirty-two times more distensible than is the human thoracic aorta. The bulbar pressurevolume curve tends to have mild hysteresis, suggestive of stress-relaxation (Landowne & Stacy, 1957). The rapid and precise return of the bulbus to its initial pressure of 0 mm Hg upon withdrawal of volume increments that amounted cumulatively to 2000 per cent of its initial volume demonstrates the marked resilience, or elasticity, of the bulbar wall. With stains specific for elastic tissue, the bulbus stains much more intensely and extensively than does the human aorta. The bulbar elastic tissue, like that of mammals, has fluorescence, which appears blue-white without, and yellow-green with, the 470 rnp barrier filter present. Elastase destroys the abilities of the bulbar media to stain with orcein and to fluoresce, confirming that these features of the bulbus are due entirely to its elastic tissue. Bulbar fluorescence is probably due to desmosine and isodesmosine, which are amino acids serving as crosslinks in mammalian elastic tissue and responsible for its fluorescence (Partridge et al., 1963). Carp bulbar and ventral aortic elastic tissues differ from mammalian elastic tissue in their coloration by hematoxylin and eosin, Masson trichrome and PTAH. The present staining results observed with human aortic elastic tissue correspond to those reported by Gillman et al. (1955). Bulbar elastic tissue is not concentrated in laminae, as it characteristically is in the carp ventral aorta and mammalian aorta, but is composed, instead, of amorphous and fibrous constituents.
STUDY OF THE TBLBOST BULBUS ARTERIOSUSIN THE CARP
707
Most of the carp bulbar elastic tissue is soluble in hot O-1 N NaOH, as is that of Lophius piscatoriu.s (Lansing, 1959), and in warm concentrated formic acid. In striking contrast, human and canine aortic elastic tissues are not dissolved by either treatment. Although, histologically, the carp ventral aorta appears to contain less elastic tissue than does the carp bulbus, the ventral aorta yields more elastic tissue residue upon extraction than does the bulbus, albeit far less than does human proximal aorta. Thus, the carp appears to have two biochemical variants of arterial elastic tissue. One is concentrated in the bulbus arteriosus and is soluble in alkali and acid. The other is found in the ventral aorta (possibly together with the “bulbar” variant) and, like mammalian elastic tissue, is alkaliand acid-insoluble. With hot alkali extraction, the ventral aorta of the shark yields 3145 per cent elastic tissue residue (Lander, 1964, cited by Satchell, 1971), which is comparable to that of mammalian proximal aorta but about three- to fivefold that of the carp ventral aorta. Thus, in addition to having an actively contracting conus arteriosus and to lacking the elastic bulbus arteriosus, the shark differs from the teleost in having a larger amount of alkali-insoluble elastic tissue in its ventral aorta. Whether or not the carp ventral aorta contains as much elastic tissue as the shark aorta does, but with most of it alkali-soluble, is unknown. Recently, the elastic tissue of the aortic media in mammals has been shown to be synthesized by adjacent smooth muscle cells (Ross, 1971; Ross & Klebanoff, 1971). The bulbar media has plenty of smooth muscle cells, which abut against the elastic tissue and may synthesize it. It is possible that, when properly stimulated, these cells can also change the tone of the bulbar wall, perhaps by mechanisms like those suggested for mammalian elastic arteries (Bader, 1963). Such a contractile action may be difficult to show. Arndt et al. (1971) found that norepinephrine infusion in vivo did not decrease the diameter or distensibility of the canine thoracic aorta or human carotid artery. These arteries, like the bulbus, are predominantly elastic, rather than muscular. It appears likely that the smooth muscle of the bulbar media-whether or not capable of affecting bulbar tone-is there primarily to produce elastic tissue. Acknowledgements-We are grateful to the John G. Shedd Aquarium and its Director, Mr. William Braker, to Drs. Edwin F. Hirsch and Calixto Maso of the Pathology Department, Columbus-Cuneo Medical Center, to Drs. Keen Rafferty, Jr. and David Aylward and to the Illinois Conservation Department and to Mr. William Harth for their generous and invaluable help and encouragement. We thank Mrs. Julia Bulota, Miss Lucille L. Whitworth and Mr. Don Taylor for their excellent technical assistance and Mrs. Alyce V. Bode for her able secretarial help. REFERENCES ABRAHAMA. (1969) Microscopic Innerwation of the Heart and Blood Vessels in Vertebrates including Man, pp. 7-22. Pergamon Press, Oxford. ARMED FORCRS INSTITUTEOF PATHOLOGY (1949) Manual of Histologic and Special Staining Technics, 2nd Edition. Blakiston, New York. ARNDT J. O., STBGALL H. F. & WICKE H. J. (1971) Mechanics of the aorta in Go: a radiographic approach. Circulation Res. 28, 693-704. AYRR J. P. (1964) Elastictissue.In International Review of Connective Tissue Research (Edited by HALL D. A.),Vol. 2, pp. 33-100. Academic Press, New York.
708
J. HAMILTONLIGHT ANDWILLARD S. HARRIS
BADERH. (1963) The anatomy and physiology of the vascular wall. In Handbook of Physiology; Circulation (Edited by HAMILTONW. F. & Dow P.), Sect. 2, Chapt. 26, pp. 865889. Am. Physiol. Sot., Washington, D.C. BURTONA. C. (1954) Relation of structure to function of the tissues of the wall of blood vessels. Physiol. Rev. 34, 619-642. DEMPSEYE. W. & LANSINGA. I. (1954) Elastic tissue. Int. Rev. CytoZ. 3, 437-453. GILLMANT., PENN J., BRONKSD. & Roux M. (1955) Abnormal elastic fibers. Archs Path. 59, 733-749. GORE I. & LARKEY B. J. (1960) Functional activity of aortic mucopolysaccharides. J. Lab. clin. Med. 56, 839-846. HALLOCKP. & BENSON I. C. (1937) Studies on the elastic properties of human isolated aorta. J. clin. Invest. 16, 595-602. HASS G. M. (1942) Elastic tissue-I. Description of a method for the isolation of elastic tissue. Archs Path. 34, 807-819. HIRSCH E. F., KAISER G. C., BARNERH. C. & COOPER T. (1970) The innervation of the feline heart. In The Innervation of the Vertebrate Heart (Edited by HIRSCH E. F.), Chapt. 6, pp. 64-79. Charles C. Thomas, Springfield. JOHANSENK. (1962) Cardiac output and pulsatile aortic flow in the teleost, Gadus morhua. Comp. Biochem. Physiol. 7, 169-174. JOHANSENK. & MARTIN A. W. (1965) Comparative aspects of cardiovascular function in vertebrates. In Handbook of Physiology; Circulation (Edited by HAMILTONW. F. & Dow P.), Sect. 2, Chapt. 73, pp. 2583-2614. Am. Physiol. Sot., Washington, D.C. LANDOU~NE M. & STACY R. W. (1957) Glossary of terms. In Tissue Elasticity (Edited by REMINGTONJ. W.), pp. 191-201. Am. Physiol. Sot., Washington, D. C. LANSINGA. I. (1959) Elastic tissue. In The Arterial Wall (Edited by LANSINGA. I.), Chapt. 5, pp. 136-160. Williams & Wilkins, Baltimore. LILLIE R. D. (1965) Histopathologic Technic and Practical Histochemistry, 3rd Edition, p. 552. Blakiston, New York. LOWRY 0. H., GILLIGAND. R. & KATERSKYE. M. (1941) The determination of collagen and elastin in tissues, with results obtained in various normal tissues from different species. r. biol. Chem. 139, 795-804. MOTT J. C. (1950) Radiological observations on the cardiovascular system in Anguilla anguilla. J. exp. Biol. 27, 324-333. NASSART. K. & SHANKLINW. M. (1961) Simplified procedure for staining reticulum. Archs Path. 71, 611-614. PARSONSC. W. (1930) The conus arteriosus in fishes. Q.Jl microsc. Sci. 73, 145-176. PARTRIDGES. M., ELSDEN D. F. & THOMASJ. (1963) Constitution of the cross-linkages in elastin. Nature, Lond. 197, 1297-1298. PEARSEA. G. E. (1968) Histochemistry, Vol. I, pp. 214-247. Little, Brown & Co. Boston. RODRIGUEZ J. & DEINHARDTF. (1960) Preparation of a semipermanent mounting medium for fluorescent antibody studies. Virology 12, 316-317. Ross R. (1971) The smooth muscle cell-II. Growth of smooth muscle in culture and formation of elastic fibers. r. Cell Biol. 50, 172-186. Ross R. & KL.EBANOFFS. J. (1971) The smooth muscle cell-I. 1n vivo synthesis of connective tissue proteins. J. Cell Biol. 50, 159-171. SACHARL. A., WINTER K. K., SICHER N. & FRANKELS. (1955) Photometric method for estimation of elastase activity. Proc. Sot. exp. Biol. Med. 90, 323-326. SATCHELLG. H. (1971) Circulation in Fishes. Cambridge University Press. SNEDECORG. W. (1956) Statistical Methods Applied to Experiments in Agriculture and Biology, 5th Edition. Iowa State University Press, Ames, Iowa. VONSKRAMLIKE. (1935) uber den kreislauf den fischen. Ergebn. Biol. 11, l-130. Key Word Index-Bulbus arteriosus ; teleost ; carp ; ventral aorta; elastic tissue; elastase; elastic tissue solubility; elasticity; Cyprinus curpio.
Physiol., 1973, Vol. 46A, pp. 699 to 708. Pwgamon Press. Printed in Great Britain
THE STRUCTURE, COMPOSITION AND ELASTIC PROPERTIES OF THE TELEOST BULBUS ARTERIOSUS IN THE CARP, CYPRINUS CARPIO*
J. HAMILTON
LICHT
and WILLARD
S. HARRIS
Abraham Lincoln School of Medicine, University of Illinois at the Medical Center, Department of Medicine, Section of Cardiology, Chicago, Illinois 60612, U.S.A. (Received 5 January 1973) Abstract-l. Pressure-volume studies show that the bulbus arteriosus of carp (Cyprinus c&o) is extremely distensible and resilient. 2. Studies with specific elastic tissue stains, fluorescence and elastase show that the bulbus has abundant elastic tissue. 3. Bulbar elastic tissue differs from that of mammalian aorta in its coloration by non-specific stains, lack of lamellar distribution and marked solubility in hot 0.1 N NaOH and warm 89% formic acid. 4. Smooth muscle cells in the bulbus probably synthesize its elastic tissue.
INTRODUCTION THE BULBUS ARTERIOSUS
of the teleost is a thick-walled chamber that connects the single cardiac ventricle to the ventral aorta. Because of its central location in the single circulation of the fish, the bulbus can regulate the impedance faced by the ejecting ventricle and the pressure and flow occurring in the systemic arteries and in the gill capillaries. During systole the teleost ventricle ejects its contents into the bulbus arteriosus, which distends. During diastole the ventriculobulbar valve is closed, but forward flow into the ventral aorta continues as the bulbus contracts down to its previous size (Mott, 1950). The bulbar maintenance of forward flow during ventricular diastole is important in teleosts because of their long diastolic periods. The bulbus also attenuates peak systolic intraventricular pressure, thereby protecting the delicate gill capillaries from excessive distention and possible rupture. Because it buffers the otherwise wide swings of systolic and diastolic aortic pressure, the bulbus has been described as a “windkessel” (von SkrarnIik, 1935) or “pressure chamber” (Johansen & Martin, 1965). Despite the prominence and well-recognized importance of the bulbus in the teleost circulation (Johansen, 1962; Satchell, 1971), the structure, composition and elastic properties of the bulbar wall have not been clearly defined. l This investigation was supported in part by American Heart Association grants, U.S. Public Health Service Research Grants Nos. HE14412-02 and lT12-HL05879 and a U.S. Public Health Service general research support grant.
699
700
J. HAMILTON LICHT AND WILLARD S. HARRIS
Strictly, in physical terms, elasticity means resistance to deformation. More commonly, and in biology, elasticity denotes a combination of stretchability, i.e. low resistance to deformation, and complete reversibility of deformation upon removal of the deforming force, or stress (Burton, 1954; Landowne & Stacy, 1957). In this common usage, the bulbus is highly elastic. The elasticity of the bulbus has not previously been quantitated, its structure and histological staining characteristics have received but brief mention (Parsons, 1930; Abraham, 1969), and only Lansing (1959), in a terse comment on the bulbus of the anglerfish, Lophius piscatorius, has discussed the similarity and dissimilarity of elastic tissue of the bulbus to that of mammals. We have, therefore, investigated the stress-strain properties of the bulbar wall by pressure-volume studies, documented its histological and tinctorial characteristics using several different stains, determined the effects produced by elastase on two known characteristics of elastic tissue-its fluorescence and affinity for orcein (Ayer, 1964) and quantitated the amount of elastic tissue extractable from the bulbus by conventional extraction methods (Lowry et al., 1941; Hass, 1942). Because the carp (Cyprinus carpio) is an easily accessible and common fresh-water teleost, its bulbus arteriosus was studied. When appropriate, comparison studies of carp ventral aorta, human and canine proximal aortae, and elastic tissue from bovine ligamenturn nuchae were also done.
MATERIALS
AND
METHODS
Carp, Cyprinus curpio, from the upper Mississippi River were kept for several months in a continuously aerated, 6000-gal holding tank at lo-15°C and were fed chopped smelt and horse heart strips twice weekly. For about 2 weeks before study they were kept in our laboratory in a continuously filtered and aerated tank, which was equipped with a cooling unit (Living Stream Model LS-700, Frigid Units, Inc., Toledo, Ohio) that maintained water temperature at 20°C. Human proximal aortae were obtained at the post-mortem examination of three women, 39, 58 and 63 years old. Proximal aortae were excised, immediately after death, from pentobarbital-anesthetized dogs killed by transthoracic intracardiac injection of 100 cm3 air. Elastic tissue fragments from bovine ligamentum nuchae (Schwarz/Mann, Orangeburg, N.Y.) and swine elastase (65 units/mg, code ESFF, Worthington Biochemical Corp., Freehold, N.J.) were purchased. One unit of elastase solubilizes 1 mg elastin in 20 min under specified assay conditions (Sachar et al., 1955). Statistical analysis was done with Student’s t-test (Snedecor, 1956).
Pressure-volume
studies
Six carp, weighing 0.8-l .2 kg, were killed by a crushing blow to the head. Through an incised triangular flap of the ventral wall cephalad to the pectoral fins, the pericardium was cut and the ventricle, bulbus and ventral aorta excised as a unit. Bisection of the ventricle perpendicular to its long axis exposed the ventriculobulbar orifice. Through this orifice, the bulbar and ventral aortic lumina were flushed by syringe three times with 5 ml of carp Ringer solution, which contained (in mM/l.) Caa+, 2.3 ; Naf, 156; Kf, 3.1; and Cl-, 160. The solution was buffered at pH 7.5 with NaH,PO, and Na,HPO, and, as determined with a freezing-point-depression osmometer (Advanced Instruments Co., Newton Highlands, Mass.), had an osmolality of 301 mOsmoles/l.
STUDY
OF THE
TELEOST
BULBUS
ARTEXIOSUS
IN
THE
CARP
701
The ventriculobulbar orifice was closed by a silk ligature tied tightly around the ventriculobulbar groove. The blunt tip of a lo-gauge, stainless steel needle was passed retrograde through the ventral aorta into the bulbar lumen and secured in place with a silk ligature. The needle hub was attached to a stainless steel Y-adaptor. A 5-ml syringe, connected by a three-way stopcock to a second arm of the adaptor, was used for intrabulbar injections and withdrawals. The tip of a saline-filled polyethylene catheter (length 8 cm, id. O-58 mm) was passed through the third arm of the adaptor and the needle and into the bulbar lumen. The catheter hub, which closed off the orifice of the Y-adaptor, was attached directly to a Statham P23Db strain gauge transducer. The transducer, lixed horizontally level with the catheter tip, was connected to the SGM-2 pressure amplifier channel of a photographic recorder (Electronics for Medicine, White Plains, N.Y.). Full-scale pressure on the recording was 100 mm Hg and paper speed was 5 mm/set. The bulbus was suspended in a bath of oxygenated carp Ringer solution at 22°C. Before study air was evacuated from the bulbar lumen, needle and Y-adaptor by withdrawing the plunger of the empty syringe attached to the three-way stopcock until resistance permitted no further withdrawal. The stopcock was closed toward the Y-adaptor, sealing the evacuated chamber. The syringe was replaced with a 5-ml syringe filled with carp Ringer solution, which was injected until intrabulbar pressure was 0 mm Hg relative to the atmosphere. A trial consisted of adding to the intrabulbar volume at 0 mm Hg, which measured approximately 0.1 ml, five consecutive injections of 0.4 ml oxygenated carp Ringer solution, for a cumulative injectate of 2 ml, followed by five consecutive withdrawals of 0.4 ml solution each. Each step increment or decrement of volume was made in 1 set, and the bulbus was kept at the new volume for another 3-4 set before the next step change was begun. Each of the six bulbi received six to twelve trials for a total of fifty-one trials. Histologic studies Bulbi arteriosi and ventral aortae, freshly excised from two carp, and one human proximal aorta were fixed for 12 hr in 10% neutral formalin and embedded in paraffin. Nuchal elastic tissue fragments were parafhn-embedded both with and without a previous 12 hr formalin fixation. Paraffin sections, 6 /J thick, from each of the five kinds of tissue preparations were simultaneously stained in the same tray. Stains used were hematoxylin and eosin, Gomori and Masson trichrome for connective tissue, Bielschowsky silver impregnation for reticulin and nerve fibers, alcian blue for acid mucopolysaccharides and seven elastic tissue stains: Fraenkel orcein, Taenzer-Unna orcein, Weigert resorcein fuchsin, Verhoeff iron hematoxylin, Gomori aldehyde fuchsin, Gallego iron fuchsin and Mallory phosphotungstic acid hematoxylin (PTAH). Staining methods were done as described by the Armed Forces Institute of Pathology Manual of Histologic and Special Staining Technics (1949) except for the two orcein stains (Lillie, 1965) and the silver impregnation (Nassar & Shanklin, 1961; Hirsch et al., 1970). The effects of elastase on staining with orcein and on fluorescence were assessed in frozen sections, 12 p thick, of carp bulbus arteriosus and ventral aorta and canine proximal aorta placed on glass slides. One set of sections was immediately fixed in 10% formalin. The second set was layered over by pipette with 0.1 mg elastase, dissolved in 0.1 ml of 0.2 M Tris buffer at pH 8.8 (Sachar et al., 1955), and was placed in 50-ml plastic syringes, which were laid flat in an oven. Evaporation of the elastase solution was inhibited by the addition of 5-10 ml water to the bottom of the syringes, which were then sealed. After 30-min incubation at 37”C, the sections were gently rinsed with distilled water to remove enzyme and solubilized protein and fixed in formalin. Both the untreated and elastase-treated sets were stained by the Fraenkel orcein method. Other sections from both sets were mounted, unstained, between a glass slide and cover-slip with a polyvinyl alcoholbuffered saline-glycerol medium (Rodriguez & Deinhardt, 1960), and were examined for fluorescence with a Zeiss fluorescence microscope equipped with an immersion condenser
702
J. HAMILTON LICHT AND WILLARD S. HARRIS
(l-2/1.4 N.A.). When nuchal elastic tissue fragments were examined with two BG12 exciter filters (spectrum of transmission 320-510 rnp) present, the fragments fluoresced a brilliant blue-white. However, when this fluorescence was passed through a barrier filter that excluded frequencies below 470 rnp, nuchal elastic tissue fragments fluoresced a yellow-green. In the present study this yellow-green fluorescence was used to identify elastic tissue.
Extraction studies Tissues were minced with scissors and homogenized in S-10 ml of 95% ethanol in a The paste was transferred to 50 ml Pyrex Waring blendor until they were paste-like. centrifuge-tubes, refluxed for 30 min over steam in 40 ml of 95% ethanol and centrifuged at 1750 g, and the supernatant was decanted. Ethanol refluxing, centrifugation and decantation were repeated. After an ether rinse and oven-drying at 95°C for 24 hr, samples of the defatted-dried tissues were weighed. Defatted-dried bulbus arteriosus, ventral aorta and cardiac ventricle from carp, human proximal aorta and canine proximal aorta were treated by the quantitative elastin extraction method of Lowry et al. (1941), which is based on the insolubility of elastic tissue and the solubility of other tissue constituents in hot alkaline solution. Tissue samples, weighing 0.18-1.90 g, were mixed with 40 ml of 0.1 N NaOH in clean 50 ml Pyrex centrifuge-tubes, heated for 10 min at 95°C with intermittent stirring in a boiling water-bath, centrifuged for 5 min at 1750g and decanted. The precipitate was again heated for 10 min in 40 ml of 0.1 N NaOH and the mixture was centrifuged and decanted. After three centrifugerinses-each consisting of a rinse with distilled water, centrifugation for 5 min at 1750g and decantation-the precipitate was oven-dried for 24 hr at 95”C, and the resulting residue was weighed. The residues from the bulbus and human proximal aorta were examined for fluorescence. The percentage of defatted-dried tissue weight that is residue was determined as 100 times the weight of residue/weight of defatted-dried tissue. The percentage of residue weight due to inorganic constituents was determined either by incubation in dilute (0.1 N) formic acid for 8 hr at 22°C which dissolves inorganic elements, such as calcium and phosphorus (Dempsey & Lansing, 1954), or by ashing over an open flame in predried porcelain crucibles. Either the weight lost with 0.1 N formic acid or the weight of the ash was taken as the weight of the inorganic constituents. Hass (1942) reported that elastic tissue was quantitatively extracted from other tissue constituents with warm concentrated (89%) formic acid. Defatted-dried carp bulbus arteriosus, carp ventral aorta and human proximal aorta were incubated for 72 hr in 890,‘, formic acid (1 ml/5 mg tissue) at 45” C in sealed Pyrex flasks. After repeated centrifugerinses with distilled water in clean Pyrex centrifuge-tubes, the tissues were dried for 24 hr at 95” C and the resulting residues were weighed and examined with the fluorescence microscope. RESULTS
Pressure-volume
studies (Fig. 1)
After each step increase of volume, intrabulbar pressure quickly declined from the immediate postinjection level. During the first second postinjection, the pressure fell on average approximately 0.5 mm Hg and remained relatively stable over the next 2-3 sec. The intrabulbar pressures used for the graph in Fig. 1 were measured 3 set after the step change in volume. Data for each bulbus were taken as the mean of all its trials. These means are averaged, with sample N = 6, in the curve in Fig. 1, which relates pressure to the changes in intrabulbar volume for the group. With either increments or
STWXW OF THE TELEOST IBULBUS ARTERXQSUS IN THE CARP N=6
FIG,
1.
7a3
CARP
The re&t~on of intrabutbatr pressure to c-s in intrabdbar volume. Iatrabulbar volume at 0 mm Hg averaged O-1 ml.
decrementsof v~~~rne~ the curve is steepestbetween 0 and O-4ml changes in vohmie. Thereafter, the curve is more gradua& with pressure changing an average -t_S.E, of 4.8 + 0-4s mm Hg per O-4 ml change oE volume. Hysteresis is present but is not significant at the 5 per cent level at any change in volume.
The w& of the n~n~tended bulbus has three distinct coats {Fig. 2)_ The outer layer, or adventitia, which is very thin, is composed of loose fibrous connective tissue, primarily colIagen, and contains medium-sized arterioles, venules and lymphatic vessels, occasional smooth muscle fibers, rare small. nerve bundles, and smaII amounts of fat. The inxrer coat, or intima, is in some areas limited to an cndothelial lining of cuboid or Bat cells but, in other areas is a thin spongy layer, which is composed p~ar~~y of connect&e tissueS is rich in nerve fibers and contains many small capillary&ke channels. The tunica media, occupying more than 90 per cent of the unstretched bulbar wall, is much thicker in proportion to the vessel wall than is the media of mammalian elastic arteries. There is much coaaective tissue throughout the bulbar media, which also contains a circumferential& arranged layer of ~rn~~ct smooth muscler and a thicker, Ioosefy arranged internal layer of ~terdig~~~ smooth rnzfscfe far&&s. Finger-auks projections of adventitia carry vasa vasorum to the compact external smooth muscle layer of the media. In addition to having a small amount of collagen, colored blue with Masson trichromq the bulbar media is extraordinarily rich in tissue that stains strongly with or&n (Fig, 3)* Weigert resorcein fuchsin, VerhoeE iran hernato~~~~ Gallego iron fuchsin and Gomori aldehyde fuchsin, which are stains with some
704
J. H.&MILTON LICHT
AND
WILLARD S. HARRIS
specificity for elastic tissue (Dempsey & Lansing, 19.54; Pearse, 1968). These stains impart the same color to the elastic tissue, whether in the media of the carp bulbus, ventral aorta, human proximal aorta or bovine ligamenturn nuchae. Each elastic tissue stain colors the bulbus much more deeply and extensively than it does the media of the human proximal aorta or the carp ventral aorta. In contrast, the bulbar elastic tissue differs from that of mammalian specimens in its coloration by other stains. The bulk of elastic tissue in the carp bulbus and ventral aorta stains light green with Masson trichrome and does not stain with PTAH or eosin. The bovine nuchal and human aortic elastic tissues stain red with Masson trichrome, deep blue with PTAH and pink with hematoxylin and eosin. The bulbar media does contain a few fine eosinophilic fibers and a few 0 verall, however, the bulbar media is fine fibers that stain blue with PTAH. richly basophilic with hematoxylin and eosin, while the media of the human Owing to its content of acid mucopolysaccharides proximal aorta is eosinophilic. (Gore & Larkey, 1960), the media of human proximal aorta, except for its elastic laminae, stains richly blue with alcian blue. In contrast, the carp bulbs and ventral aortic media stain only slightly blue with alcian blue. Bielschowsky silver impregnation, with and without counterstaining by the shows that reticulin and nerves are elastic tissue stain, Gallego iron fuchsin, present, particularly in the intimal and subadventitial regions of the bulbus, and that these argyrophilic elements are clearly different from the bulbar elastic tissue. The formalin-fixed fragments of nuchal elastic tissue had the same staining characteristics as the nonfixed nuchal fragments. When frozen sections were examined with the fluorescence microscope, elastic tissue of the carp bulbus and ventral aorta, like the nuchal elastic tissue and the elastic laminae of the canine proximal aorta, fluoresced vividly yellow-green. Application of elastase to the frozen sections of all three tissues reduced the widespread, deep yellow-green fluorescence to a small amount of yellow granular debris (Fig. 4) and abolished the staining response to orcein. Medial elastic tissue in the bulbus appears to differ morphologically from that In man and dog, the elastic tissue is in the human and canine proximal aortae. concentrated in laminae, from which fine elastic tissue fibers emanate. The media
FIG. 3A. Part of the internal layer of bulbar media stained for elastic tissue, which appears black here. The light-grey region (lower left) is collagen-rich ventriculobulbar valvular tissue, included here for contrast. (a) Internal layer of bulbar media. Taenzer-Unna orcein stain (X 112). B. (b) Bulbar lumen. (c) Valvular tissue. Note the dark staining elastic laminae. Elastic tissue in human proximal aorta. (a) Media. (b) Intima. Taenzer-Unna orcein ( x 112). The carp bulbus in (A) and the human proximal aorta in (B) were stained simultaneously in the same tray and were photographed and developed under identical conditions. The bulbus stains more intensely and extensively with specific elastic tissue stains than does the human aorta. C. Higher magnification of the valvular region shown in (A). The arrows at (a) point to the amorphous elastic tissue. (b) Coarse elastic fibers. The arrows at (c) point to fine elastic fibers. ( x 440.)
FIG. 2A. Part of a cross-section of the nondistended bulbar wall with the pericardium removed. (a) Bulbar adventitia. (b) External layer of bulbar media containing compact smooth muscle. (c) Internal layer of bulbar media containing more loosely arranged smooth muscle. (d) Bulbar intima. (e) Bulbar lumen. (f) Blood in the bulbar lumen. (g) Ventriculobulbar groove. (h) Ventricular myocardium. Gomori trichrome ( x 20.5). B. Whorls of smooth muscle in the internal layer of the bulbar media. This stain does not show the rich elastic component of the bulbar wall. Gomori trichrome. ( x 262.)
FIG.
3
(a)
(b)
FIG. 4. Effects of elastase on fluorescence of elastic tissue in frozen sections. Fluorescence appears white here. The top three panels show untreated elastic tissue, while the bottom three panels show the same tissues after 30 min incubation with elastase. (a) Canine proximal aorta. Note the thick elastic laminae. (b) Carp ventral aorta with laminae. (c) Carp bulbus arteriosus. For clarity, a trabeculated area of bulbar media near the lumen is shown; the black lacunae are areas of the lumen. In all three tissues, as the lower panels show, elastase treatment reduces the fluorescence to a small amount of granular debris. (Approximately x 115.)
705
STUDYOF THBTBLBOSTBULBUSABTBRIOSUS IN THE CARP
of the carp bulbus, by contrast, is pervaded throughout by both amorphous tissue and numerous coarse and fine fibers, all of which stain deeply with the elastic tissue stains. Each smooth muscle cell of the bulbus appears to be enveloped by a mantle of elastic tissue. Unlike the bulbus, the carp ventral aorta contains elastic tissue laminae, which resemble closely those found in the human and canine proximal aortae. To a distinctly lesser extent than in the bulbus, the carp ventral aortic media is also pervaded by nonlamellar elastic tissue. Extraction
studies
As Table 1 shows, the percentage of defatted-dried tissue weight remaining as residue after 0.1 N NaOH extraction was (mean+ S.E.) 0.8 f 0.12 per cent for carp bulbus arteriosus, 8.9 _+2.31 per cent for carp ventral aorta, 39.6 + 1.45 per cent for human proximal aorta and 32.6 per cent for canine proximal aorta. Defatted-dried carp ventricular tissue (1.90 g) had no measurable elastic tissue residue. Inorganic constituents, determined by O-1 N formic acid treatment or by ashing, averaged 47.4 per cent of carp bulbar residue, 9.8 per cent of the carp ventral aortic residue and 12.1 per cent of human proximal aortic residue. Therefore, bulbar, carp ventral aortic and human proximal aortic residue weights were multiplied by O-526, 0.902 and O-879 respectively to obtain weights corrected for TABLE ~-WEIGHT OF RESIDUEMTBACTBDWITH 0.1 N NaOH OR 89% FORMICACID
Tissue
No. of determinations
Total number of specimens
Residue weight * (%)
Residue after correction for inorganic content* (%)
O-1 N NaOH Carp bulbus arteriosus Carp ventral aorta Carp cardiac ventricle Human proximal aorta Canine proximal aorta
105 29 7 3 1
10 3 1 3 1
O-8 * 0.12f 8.9 + 2.31 0.0 39.6 f l-45 32-6
0.4 !I 0.06 8.0 f 2.07 34.8 + 1.27
89% formic acid Carp bulbus arteriosus Carp ventral aorta Human proximal aorta
7 2 3
* Percentage residue weight =
109 15 3
9.3 f 1.52 18.4 44.1 iz 4.56
weight of dried residue (mg) weight of defatted-dried tissue (mg)
x 100.
t Percentage residue weight after correction for inorganic content = per cent residue weight x the appropriate correction factor, which is given in the text. $ Mean + S.E. with the number of determinations used as the sample iV.
706
J. HAMILTON
LICHT AND WILLARD S. HARRIS
inorganic content. Corrected values, expressed as percentage residue after correction for inorganic content, are given in Table 1. Residues from bulbus and human proximal aorta, which were the only ones examined by fluorescence microscopy, fluoresced vividly yellow-green. With human proximal and carp ventral aortae, extraction with 89 per cent formic acid proceeded as described by Hass (1942). However, during centrifugerinsing of the bulbar tissue, approximately 15 ml of a translucent gel, or coagulum, which entrapped fine tissue fragments and fibers, formed and persisted despite repeated rinsing. For elimination of the gel, it was oven-dried for 24 hr at 95°C rehydrated with 40 ml of distilled water and heated for 30 min at 95°C in a boiling water-bath. After centrifugation and redrying, the samples were reweighed. The bulbar residue was 9.3 _t 1.52 per cent of the defatted-dried tissue weight (Table 1). The carp ventral aortic and human proximal aortic residues were 18.4 per cent and 44.1 k 4.56 per cent of defatted-dried tissue weight, respectively. The residues from each of the three kinds of tissues fluoresced vividly yellow-green.
DISCUSSION
In human thoracic aorta, the volume at 7.3 mm Hg pressure, which was 9 ml, was found to increase 22 per cent when pressure was raised to 33 mm Hg (Hallock & Benson, 1937). In the present study (Fig. l), intrabulbar volume (0.3 ml) at 7.3 mm Hg was found to increase 700 per cent when pressure rose to 33 mm Hg. In this range of pressures, therefore, the carp bulbus arteriosus is about thirty-two times more distensible than is the human thoracic aorta. The bulbar pressurevolume curve tends to have mild hysteresis, suggestive of stress-relaxation (Landowne & Stacy, 1957). The rapid and precise return of the bulbus to its initial pressure of 0 mm Hg upon withdrawal of volume increments that amounted cumulatively to 2000 per cent of its initial volume demonstrates the marked resilience, or elasticity, of the bulbar wall. With stains specific for elastic tissue, the bulbus stains much more intensely and extensively than does the human aorta. The bulbar elastic tissue, like that of mammals, has fluorescence, which appears blue-white without, and yellow-green with, the 470 rnp barrier filter present. Elastase destroys the abilities of the bulbar media to stain with orcein and to fluoresce, confirming that these features of the bulbus are due entirely to its elastic tissue. Bulbar fluorescence is probably due to desmosine and isodesmosine, which are amino acids serving as crosslinks in mammalian elastic tissue and responsible for its fluorescence (Partridge et al., 1963). Carp bulbar and ventral aortic elastic tissues differ from mammalian elastic tissue in their coloration by hematoxylin and eosin, Masson trichrome and PTAH. The present staining results observed with human aortic elastic tissue correspond to those reported by Gillman et al. (1955). Bulbar elastic tissue is not concentrated in laminae, as it characteristically is in the carp ventral aorta and mammalian aorta, but is composed, instead, of amorphous and fibrous constituents.
STUDY OF THE TBLBOST BULBUS ARTERIOSUSIN THE CARP
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Most of the carp bulbar elastic tissue is soluble in hot O-1 N NaOH, as is that of Lophius piscatoriu.s (Lansing, 1959), and in warm concentrated formic acid. In striking contrast, human and canine aortic elastic tissues are not dissolved by either treatment. Although, histologically, the carp ventral aorta appears to contain less elastic tissue than does the carp bulbus, the ventral aorta yields more elastic tissue residue upon extraction than does the bulbus, albeit far less than does human proximal aorta. Thus, the carp appears to have two biochemical variants of arterial elastic tissue. One is concentrated in the bulbus arteriosus and is soluble in alkali and acid. The other is found in the ventral aorta (possibly together with the “bulbar” variant) and, like mammalian elastic tissue, is alkaliand acid-insoluble. With hot alkali extraction, the ventral aorta of the shark yields 3145 per cent elastic tissue residue (Lander, 1964, cited by Satchell, 1971), which is comparable to that of mammalian proximal aorta but about three- to fivefold that of the carp ventral aorta. Thus, in addition to having an actively contracting conus arteriosus and to lacking the elastic bulbus arteriosus, the shark differs from the teleost in having a larger amount of alkali-insoluble elastic tissue in its ventral aorta. Whether or not the carp ventral aorta contains as much elastic tissue as the shark aorta does, but with most of it alkali-soluble, is unknown. Recently, the elastic tissue of the aortic media in mammals has been shown to be synthesized by adjacent smooth muscle cells (Ross, 1971; Ross & Klebanoff, 1971). The bulbar media has plenty of smooth muscle cells, which abut against the elastic tissue and may synthesize it. It is possible that, when properly stimulated, these cells can also change the tone of the bulbar wall, perhaps by mechanisms like those suggested for mammalian elastic arteries (Bader, 1963). Such a contractile action may be difficult to show. Arndt et al. (1971) found that norepinephrine infusion in vivo did not decrease the diameter or distensibility of the canine thoracic aorta or human carotid artery. These arteries, like the bulbus, are predominantly elastic, rather than muscular. It appears likely that the smooth muscle of the bulbar media-whether or not capable of affecting bulbar tone-is there primarily to produce elastic tissue. Acknowledgements-We are grateful to the John G. Shedd Aquarium and its Director, Mr. William Braker, to Drs. Edwin F. Hirsch and Calixto Maso of the Pathology Department, Columbus-Cuneo Medical Center, to Drs. Keen Rafferty, Jr. and David Aylward and to the Illinois Conservation Department and to Mr. William Harth for their generous and invaluable help and encouragement. We thank Mrs. Julia Bulota, Miss Lucille L. Whitworth and Mr. Don Taylor for their excellent technical assistance and Mrs. Alyce V. Bode for her able secretarial help. REFERENCES ABRAHAMA. (1969) Microscopic Innerwation of the Heart and Blood Vessels in Vertebrates including Man, pp. 7-22. Pergamon Press, Oxford. ARMED FORCRS INSTITUTEOF PATHOLOGY (1949) Manual of Histologic and Special Staining Technics, 2nd Edition. Blakiston, New York. ARNDT J. O., STBGALL H. F. & WICKE H. J. (1971) Mechanics of the aorta in Go: a radiographic approach. Circulation Res. 28, 693-704. AYRR J. P. (1964) Elastictissue.In International Review of Connective Tissue Research (Edited by HALL D. A.),Vol. 2, pp. 33-100. Academic Press, New York.
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