Morphological Study on Microvasculature of Left Ventricular Wall in Infant and Adult Yaks.код для вставкиСкачать
THE ANATOMICAL RECORD 293:1519–1526 (2010) Morphological Study on Microvasculature of Left Ventricular Wall in Infant and Adult Yaks Y.Y. HE,1 S.J. YU,2 Y. CUI,2* AND P. DU2 Instrumental Research and Analysis Center, Gansu Agricultural University, Gansu, China 2 College of Veterinary Medicine, Gansu Agricultural University, Gansu, China 1 ABSTRACT Observations on the microvasculature in the left ventricular wall of infant and adult yaks under light and scanning electron microscope (SEM) were presented. Moreover, the diameter of different microvasculature and the density of the capillaries in three layers of the ventricular myocardium were measured using Image Pro-Plus 5.0. The results showed that the average luminal diameter of arterioles and precapillary arterioles in adult yak’s hearts were, in most cases, larger than those in infant yaks. On the contrary, the diameters of the capillary in infant yak’s hearts were larger than those in adult yaks. The density of capillary in the myocardium of adult yak’s heart had signiﬁcantly higher values (P<0.01) than those in infant yaks. Arterioles of yak’s hearts were characterized by the bark-like structure and the impressions of endothelial cell nuclei, and it always gave rise to capillary after three to four grades of embranchment. The outer surface of capillaries cast in infant yak’s hearts was smooth, and no constrictions were found. This was different from adult yak that always had some constrictions. The capillary anastomosis of ‘‘H’’ and ‘‘Y’’ usually existed in the myocardium of both adult and infant yaks; however, those in infant yaks were not typical as in adult yaks in their shape. The peculiar arrangement of the venules in infant yak was a ‘‘baggy’’ or ‘‘bulgy’’ arrangement, while in the adult yak, they had a root-like pattern. Our ﬁndings suggest that the patterns of microvasculature in yak’s heart could be propitious to adapt better in their environment following their increase of age. Anat C 2010 Wiley-Liss, Inc. Rec, 293:1519–1526, 2010. V Key words: yak; left ventricle; microvasculature; diameter; density In previous studies, the characteristics of microvasculature in many kinds of domestic mammals have been demonstrated (Bayer et al., 2002; Grabherr et al., 2008). Some used microangiography (Speck, 2003; Djonov and Makanya, 2005; Suo et al., 2007), and some used the method of vascular corrosion casting (Krucker et al., 2006; Lametschwandtner et al., 2006). As Meyer et al. (2008) said, the combination of scanning electron microscope (SEM) and vascular corrosion casting is a powerful and best method for studying vascular morphology and architecture. Although there is a long-lasting interest in the microvasculature of heart, the ﬁne distribution and the three-dimensional arrangement of the entire microvasculature bed of heart still remain to be elucidated in C 2010 WILEY-LISS, INC. V more detail. What is more, there are no studies done where the heart’s microvasculature of the infant yak is compared with that of the adult animal. Grant sponsor: Nature Science Foundation of China; Grant number: 30960270. *Correspondence to: Y. Cui, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, Gansu 730070, China. E-mail: firstname.lastname@example.org Received 13 October 2008; Accepted 30 March 2010 DOI 10.1002/ar.21201 Published online 24 July 2010 in Wiley Online Library (wileyonlinelibrary.com). 1520 HE ET AL. Yak is one of the most important breed living in high mountain grassland at an extreme cool temperature and low oxygen content. Therefore, yak’s heart must have the stronger function to ﬁt the special environment. As in high altitude, the amount of oxygen available for animals decreases and the number of red blood cells tends to increase making the blood more viscous. It would be interesting to know whether the heart of yak that has to pump blood against a then increased resistance copes with this situation by adaption of the microvasculature of its muscular layers to keep the energy costs for its perfusion low. The present study was undertaken to observe the microvascular in ventricular wall of infant and adult yaks. Although it is reasonable to suppose that the basic pattern is likely to be similar in infant and adult yak’s hearts, there must have a maturation of the microvasculature bed from juvenile to the adult in terms of the diameter, arrangement, and conﬁguration of the microvasculature and the density of capillary. Otherwise, there are certain important age differences that must be appreciated before the results of any experimental work can be evaluated. Such information would also be useful in exploring the relationship between heart microvasculature and ambient oxygen partial pressure. MATERIALS AND METHODS Yak Twelve yaks of infant and adult ages were included in the investigation. They were purchased from small holders in Datong County of Qinghai Province. Infant yaks varied from 120 to 180 days of age and 30–50 kg of weight, and those of adult yaks were 5–8 years and 100– 200 kg, respectively. No apparent diseases were found before they were sampled. Sampling Yaks were exsanguinated, and the vascular system was injected via coronary artery. In some cases, perfusions of glutaraldehyde were made initially, followed by injection of the solution of ABS in butanone to make microcorrosion casts, and the solution of formalin (40%, p.A.) blended with black Chinese ink (vþv ¼ 1þ6) to make tissue sections, either under controlled pressure (150–200 mmHg) or hand pressure. We used three kinds of concentration of ABS solution (5% ABS in butanone, 10% ABS in butanone, 15% ABS in butanone) when we injected the heart of yak via both right and left coronary artery at the same time. At the ﬁrst stage of injection, we used 5% ABS, then 10% ABS, and last, 15% ABS. The heart that was injected with three kinds of concentration of ABS solution was kept in cold water for about 48–72 hr for complete ﬁxation, frozen fast and then the specimen was cut to 1 cm3 with a razor blade in epicardium, myocardium, and endocardium of left ventricular wall from anterior, posterior, and apex, respectively. These small specimens were placed in a solution of 30% HCl to remove the soft tissue. The specimens remained in this solution for a period of 3–6 days and were then ﬂushed with tap water to remove the residue from the cast. For a ﬁnal cleaning and removal of the residue, specimens were placed in an ultrasonic cleaner for 3 min. After complete tissue corrosion and cleaning, the casts were frozen in water, cut with a razor blade, air dried, coated with gold, and observed in an SEM. The heart injected with formaldehyde in black Chinese ink via both the right and left coronary artery, at the same time, was immersed in 5% neutral aqueous formalin for 72 hr for further ﬁxation, then sampled, dehydrated by an ascending series of alcohol, and embedded in parafﬁn wax. The tissue sections were cut from sampled blocks at 20 and 40 lm in thickness. The microvasculature was apparent when the entire microvasculature was completely injected with the solution of formaldehyde in black Chinese Ink. Measurements For quantiﬁcation of the diameter of the microvasculature, the stereopaired images were taken with SEM (Hitachi S-3400N) and were measured with its incidential software at an accelerating voltage of 20 kV and magniﬁcations ranging from 75 to 1,500 times. For quantiﬁcation of the density of capillary, the images of transverse tissue sections of the epicardium, myocardium, and endocardium, to whom the solution of formaldehyde in carbon black ink were injected, were taken with the Olympus DP-controller 70. Five different areas (two anterior, two posterior, and one apex) in each steps of left ventricular wall from anterior, posterior, and apex were selected. In each area, 20 photos with the magniﬁcation of 100 times were taken. Finally, the number of capillary in each photos were counted with the Imageproplus 5.0. Data were subjected to one-way analysis of variance (ANOVA) between steps (endocardium, myocardium, enpicardium) and ages (adult and infant). RESULTS The Diameter and Characteristic of Arteriole The arterioles of adult yak’s hearts were characterized by the bark-like structure and impressions of endothelial cell nuclei (Fig. 1A). At this level, it was found that a single layer of plastic ‘‘strips’’ was wrapped around the outer surface of the cast (Fig. 1B). With the decrease of diameter, the number of circular strips became fewer, and in some cases, they were observed as partially torn away from the cast. Arterioles decreased in size by branching in a dichotomous or pectinate manner to form capillaries, with always two daughter vessels of equal sizes. In general, the terminal arteriole divided into two daughter capillaries, and these capillaries could pursue the same direction but often ran in opposite directions (Fig. 1C). Arterioles frequently had a distinctive and characteristic arrangement, which might be described variously as helical, bellows-like or undulating (Fig. 1D,E). In most cases, the earlier features were similar in an infant yak’s heart. The average luminal diameter of the arterioles in infant and adult yak’s hearts were 73.50 8.64 and 78.50 9.72 lm, respectively, with the ranges of 11.53–100 lm and 12.5–100 lm, respectively. The arterioles of yak’s hearts gave rise to capillary after three to four grades of embranchments (Fig. 1F). The average luminal diameter of the different grades of embranchment from the ﬁrst to fourth in the infant yaks were 85.66 4.53, 63.69 6.21, 44.38 5.90, and 35.45 6.17 lm, respectively; and those in the adults were 87.64 4.87, 69.46 6.67, 48.52 5.77, and MICROVASCULATURE OF LEFT VENTRICULAR WALL IN YAK 1521 Fig. 1. The arterioles of heart in infant and adult yaks. A: The barklike structure (quadrate-boxed area) and impressions of endothelial cell nuclei (circular boxed area). The asterisk marks a transversely cut vessels. B: Single layer of ‘‘plastic strip’’ wrapped around the surface of the cast (asterisk). C: Arterioles decreased in size by branching in a dichotomous or pectinate manner to form capillaries. A, arteriole; C, capillary. D,E: Arterioles were described variously as helical, bellow- like, or undulating under both light and scanning electron microscope (arrow). Boxed areas mark blind ending vessels with rounded endings. F: The arterioles of yak’s hearts common give rise to capillary after three to four grades of embranchment. A, arteriole; A1,2,3,4, the ﬁrst-, second-, third- and fourth-grade arteriole; Pa, precapillary arteriole; C, capillary. 30.45 5.44 lm, respectively. According to those measurements, the average luminal diameter of arterioles in adult yak’s hearts were, in most cases, larger than those in infant yaks, except that in the fourth grade of embranchment. 16.24 2.27 lm, respectively, with the ranges of 11.53– 15.49 and 12.50–19.99 lm, respectively. At the level of the precapillary arteriole, the most striking feature in the precapillary arteriole was a long series of constrictions, which were evident at all of the site sampled both in infant and adult yaks. In general, the constrictions completely encircled the luminal cast; however, they were occasionally irregular or semicircular in their appearance. In an adult yak, there was a marked change in luminal diameter of precapillary arteriole, giving it a distinctive cone-shaped appearance (Fig. 2A). The Diameters and Characteristics of Precapillary Arterioles The average luminal diameters of precapillary arterioles in infant and adult yak’s hearts are 13.20 2.35 and 1522 HE ET AL. the capillaries usually had some constrictions (Fig. 3F). The density of capillary in epicardium, myocardium, and endocardium in infant yak’s hearts were 1,674 243/ mm2, 2,407 328/mm2, and 1,489 337/mm2 and those in adult yaks’ hearts were 1,864 179/mm2, 2,528 263/mm2, and 1,636 235/mm2, respectively. The density of capillary in myocardium were signiﬁcantly higher than those in epicardium and endocardium either in infant or adult yak’s hearts (P<0.01), and the density of capillary in myocardium of adult yak’s heart had signiﬁcantly higher values (P<0.01) than those in infant yak. Venules The peculiar arrangement of the venules was a striking feature of the microvasculature. A collecting venule looked quite different from a terminal arteriole. The venous capillaries were gathered from the capillary sheets into venule that was commonly oriented in a direction perpendicular to that of the capillaries (Fig. 4A). The capillaries came from all directions along the muscle ﬁbers, made a sweeping curve to join with each other, and rapidly formed a venule (Fig. 4B). The capillaries frequently were spaced at regular intervals when entering the venules and did so only on the axis parallel to the muscle ﬁber. This unusual appearance of the venules and small veins had been called the ginger or turniproot arrangement and sometimes was quite spectacular in adult yak (Fig. 4C,D). But in infant yak, it looked like a pockety arrangement (Fig. 4E,F). DISCUSSION Fig. 2. The precapillary arterioles of heart in infant and adult yaks. A: A long series of constrictions (arrowheads) and a distinctive coneshaped appearance in the cast of precapillary arteriole in an adult yak (boxed area). Asterisks mark blind-ending incompletely ﬁlled vessels. A, arteriole; Pa, precapillary arteriole; B: Long series of constrictions (arrowheads) with no distinct decrease in the diameter of the precapillary arteriole of the infant yak. capillary in each case of adult yaks was considered to begin at a point where the luminal diameter became uniform, while that was not evident in infant yak (Fig. 2B). Capillary The average diameter of capillary in infant and adult yak’s hearts were 8.57 1.29 and 6.57 2.28 lm, respectively, with the ranges of 5.75–11.53 and 6.25– 12.50 lm, respectively. The diameters of the capillary in infant yak’s hearts were larger than those of adult yaks. Generally, the precapillary arterioles gave rise to a number of capillary, but occasionally, a capillary would originated from an arteriole. Capillaries were in parallel with each other in myocardium (Fig. 3A,B), while their arrangement were irregular in endocardium and epicardium (Fig. 3C). It was clearly found that the capillaries lay within the fascicles and did not cross between their layers. The capillary anastomosis of ‘‘H’’ and ‘‘Y’’ usually existed in the myocardium of both adult and infant yaks (Fig. 3A,B,D); however, those in infant yaks were not typical as in adult yaks in their shape (Fig. 3E). In addition, the outer surface of capillaries casts in infant yaks were smooth, and no constrictions were found in the plastic cast of capillaries (Fig. 3D), while in adult yaks, To examine the sequential morphological changes of the microvasculature, heart’s samples from different age group should be included. However, the fetus heart was too small to inject the solution into the whole microvascular system successfully, so only the samples from infant and adult yaks were chosen in this investigation. Several methods for injections of the microvasculature of the heart with various materials were employed in previous studies by many scientists. Filling vessels with special materials is an established procedure and many different compounds have been reported so far. Besides the well-known polymerized methyl methacrylate resin (Anderson and Anderson, 1978; Gannon, 1981; Ohtani and Gannon, 1982), mostly PU4ii (Krucker et al., 2006) and Mercox-Cl-2B (Kachlik et al., 2002, 2007; Lametschwandtner et al., 2004, 2006; Minnich et al., 2007) have been historically used. In the present study, we found that the combination of the method using the solution of ABS in butanone for microcopists with the method using the solution of formaldehyde in black Chinese ink for tissue sections would provide a satisfactory way for both planar and three-dimensional observations. ABS is an amorphous thermoplastic blend, with the recipe of 15%–35% acrylnitrile, 5%–30% butadiene, and 40%–60% styrene. Generally, ABS has satisfactory stiffness and dimensional stability, glossy surface, and is easy to machine. The molding miniﬁcation of this kind of ABS is 0.4%–0.7%, and the same blend properties has the same shrinkage behavior. Therefore, we ignored the molding miniﬁcation when the diameter of the microvasculature cast was measured in this study. According that different concentrations of ABS have different viscosity MICROVASCULATURE OF LEFT VENTRICULAR WALL IN YAK 1523 Fig. 3. The capillaries of heart in infant and adult yaks. A,B: Capillaries in myocardium are in parallel with each other. C: Irregular arrangement of capillaries in the endocardium. D: ‘‘H’’ (white arrow) and ‘‘Y’’ (black arrow) capillary anastomoses are found frequently in the myocardium of adult yak. E: ‘‘H’’ (white arrow) and ‘‘Y’’ (black arrow) capil- lary anastomoses in infant yak. Note that diameters of capillaries are not uniform, and anastomoses differ from that in adult yak. Boxed areas mark blind ending vessels with rounded endings. F: Capillaries of the heart in adult yaks with characteristic constrictions (arrowheads). and ﬂuidity, and different vasculature in the heart has different diameter, we used three kinds of concentration of ABS solution when we injected the heart of yak, they were 5% ABS in butanone, 10% ABS in butanone, and 15% ABS in butanone, respectively, and their injection order was 5% ﬁrst, 10%, and 15% last. Because the diameter of capillary is the smallest in all of the vasculature of heart, so we used 5% ABS ﬁrst, which could easily pass through the capillary and cast the whole capillary bed. And for the same reason, because of the high viscosity and low ﬂuidity of 15% ABS, we used it at the last stage of injection, which could ﬁll the coronary artery completely and prevent outﬂow of the solution that we had injected in the heart like a stopper. In addition, we injected the heart of yak via both right and left coronary artery at the same time. This method was propitious to keep the balance of injection pressure in the vasculature, avoid the rupture of blood vessel availably, and get the perfect effect of microvasculature injection at last. Also, we found that the hand pressure that can offer a sensitive pressure and gave the best results were better than controlled pressure, in spite of there being no manometric control of injection pressure, and it was the same as the method of Ohtani and Gannon (1982). Impressions into the plastic compound produced by nuclei of endothelial cells have been described previously 1524 HE ET AL. Fig. 4. The venules of heart in infant and adult yaks. A: Orientation of venules. Note that venules (arrows) run perpendicularly to capillaries. B: The capillaries made a sweeping curve to join with each other and rapidly form venules. Asterisks mark blind-ending incompletely ﬁlled vessels. V, venules; C, capillary. C,D: Spectacular ginger or turnip-root patterns of venules in adult yak (boxed area). V, venule. E,F: The arrangement of venules in infant yak looks like a pocket (boxed area). V, venule. in the hypophyseal vessels of the toad and in the cochlear vessels of the rat even throughout the capillary network (Lametschwandtner et al., 1976; Hodde et al., 1977). Similar nuclear impression was found in the arteries and arterioles of both the canine abdominal vessels and the canine and equine intracranial vessels (Anderson and Anderson, 1978, Bayer et al., 2002). Lametschwandtner et al. (2004) also found the long endothelial cell nuclei imprints, orientated parallel with the vessel axis, and considered it as a characteristic that differentiated from venous. In this study, we still found that the nuclear impression in the cast of arterioles either in infant or adult yak’s hearts. But it was not evi- dent in the capillary portions of the casts due to the absence of the smooth muscle in the vessel walls, which, presumably, permitted relatively greater distension of the capillary. It has been suggested that the innermost layer of smooth muscle of the walls of small arterioles is arranged circularly in some areas (Anderson and Anderson, 1978), helically in others (Rhodin, 1967), and both circularly and spirally in others (Rhodin, 1978; He and Cui, 2007). We found that the same spirally arrangement in arterioles of both infant and adult yak’s hearts, with the characteristic appearance of the arteriolar casts of the heart in contracted states. We would suggest that MICROVASCULATURE OF LEFT VENTRICULAR WALL IN YAK the contraction of the smooth muscle of the vessels may produce a spring-like action of the arteriole by which blood ﬂow is effectively reduced in contraction and restored in relaxation. On the other hand, this arrangement most likely resulted from the lack of intraluminal pressure during the casting procedure. More morphological evidence is required to support this spring-action concept. The occurrence of the plastic strips overlaid around the lumen is rather a peculiar phenomenon. It has been observed in both experimental models and humans (Anderson and Anderson, 1978; Hodde 1981; Lametschwandtner et al., 2004). A considerable number of authors have related the presence of plastic strips to a corrosion defect (Schraufnagel, 1987; Christofferson and Nilsson, 1988). However, Wolff (1977) noted that localized regions of the arterioles were highly permeable to large tracer molecules such as ferritin, lanthanum, and alcian blue. It is possible that the perfusion pressure opened the tight junctions of the endothelial cells. With the lack of an internal elastic lamina, an open extracellular communication exists between the vessel lumen and the tunica media, which allows for the passage of large molecules, such as ABS in this study. Thus, the wrapping of the plastic around the ﬁlled lumen is not considered to be a replication of the smooth muscle cells but rather represents the spaces between compressed cells, and this idea is in accordance with Castenholz et al. (1982). Possibly, the frequent lack of plastic strips on vessel replicas due to the difference in pressure required to achieve the passage of material to the muscle layer. The precapillary arterioles in adult yak’s hearts were readily distinguished by the prominent long series of constrictions left by the smooth muscle cell or by the abrupt decrease in luminal diameter that gave a cone-like shape to the arteriole. These vessels correspond closely to the precapillary sphincter area and were suddenly altered drastically to that of a capillary, producing a ‘‘bottle-neck’’ effect at the level of the sphincter. The authors agree with the interpretation of Nicoll (1971) that the precapillary sphincter area is composed of a series of constrictions along the terminal twigs of the arterioles system, which may modify the ﬂow and pressure in the capillary network. The precapillary arterioles in infant yak’s hearts, however, were not found to show obvious constrictions and cone-like shape, and they could be only determined according to the diameter of their luminal cast and the context of vein. The morphological pattern of venules in infant and adult yak’s hearts was similar to those in dog (Reynolds et al., 1958) and in the work of Brown (1965) on the six domestic species. In addition, we have found the pockety arrangement in infant yak’s hearts. Compared with the arrangement of arterioles, we support a quickly reﬂux function of these microvasculature, which can better control blood pressure. Makanya et al. (2007) found that there were blind endings in the capillary corrosion cast of the developing chicken embryo lung and considered it as sprouting angiogenesis, which was responsible for the formation of the basic vascular pattern. Joachim et al. (2002) observed the presence of blind endings vessels in the primary tumors. In this study, we also observed the blind endings vessels in the heart of yak. Given the structural similarities with branching trees, it has been suggested 1525 that the growth of the cardiovascular system follows a mode in which tiny tubules in the size range of capillaries "sprout" from existing "mother" vessels. These sprouts, which form new vascular branches, then grow in length until they meet another vessel to which they connect. This process allows for the establishment of ﬂow between both preexisting vessels. It is, however, still not clear how the growth of sprouts is directed toward one another so that their fusion is possible. The present study made a serial investigation on the density of capillary in three layers of hearts in infant and adult yaks. We found that arterioles of yak’s hearts always give rise to capillary after three to four grades of embranchments, and so we did the classiﬁcation to arterioles according their arrangement and diameter of each grade arterioles. Also, we agree the idea of Gössl et al. (2003) that the arterioles have a distributing function. The distributing function of arterioles is indicated by a signiﬁcant decrease of microvasculature diameter at each bifurcation level. Moreover, it was found that the density of capillary in myocardium of adult yak’s heart had signiﬁcantly higher values than those in infant yak, while the diameter of capillary in infant yaks were larger than those in adult yaks. 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