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Veterinary Pathology Online http://vet.sagepub.com/ Blood and Serous Cysts in the Atrioventricular Valves of the Bovine Heart P. S. Marcato, C. Benazzi, G. Bettini, M. Masi, L. Della Salda, G. Sarli, G. Vecchi and A. Poli Vet Pathol 1996 33: 14 DOI: 10.1177/030098589603300102 The online version of this article can be found at: http://vet.sagepub.com/content/33/1/14 Published by: http://www.sagepublications.com On behalf of: American College of Veterinary Pathologists, European College of Veterinary Pathologists, & the Japanese College of Veterinary Pathologists. Additional services and information for Veterinary Pathology Online can be found at: Email Alerts: http://vet.sagepub.com/cgi/alerts Subscriptions: http://vet.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav >> Version of Record - Jan 1, 1996 What is This? Downloaded from vet.sagepub.com by guest on July 25, 2013 Vet Pathol33:14-21 (1996) Blood and Serous Cysts in the Atrioventricular Valves of the Bovine Heart P. S. MARCATO, C. BENAZZI, G. BETTINI, M. MASI,L. DELLA SALDA, G. SARLI,G. VECCHI, AND A. POLI Institute of Veterinary Pathology, University of Bologna, Bologna, Italy (PSM, CB, GB, LDS, GS); Unita Sanitaria Locale 24, Budrio, Bologna, Italy (MM); Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia, Sezione di Bologna, Bologna, Italy (GV); and Department of Animal Pathology, University of Pisa, Pisa, Italy (AP) Abstract. A survey of 30,907 slaughterhouse cattle (5,984 calves, 15,937 young adult, 8,986 cows) was carried out to determine the incidence of blood and serous cysts on atrioventricular valves. The cysts were classified by their content (blood/serous fluid), location (mitral/tricuspid valve), and size. Cyst wall samples were processed for histology, immunohistochemistry for factor VIII-related antigen, and transmission electron microscopy. The content of some cysts was studied by electrophoresis and biochemical and microbiologic methods. Older cows had a higher incidence (16.2%) than younger animals (1 1.5% in calves, 7.9% in steers, 6.4% in heifers), suggesting that the lesions may be acquired. Blood cysts were often present on both atrioventricular valves; serous cysts prevailed on the mitral valve. Cysts of both types were larger in older animals; serous cysts were larger than the blood cysts. Histologically, blood cysts contained fresh blood, and serous cysts were filled with a hyaline fluid devoid of cells, sterile, and biochemically similar to lymph. All the cysts were lined with endothelium, but a positive immunostaining for the factor VIII-related antigen was appreciable only in blood cysts. Ultrastructurally, the endothelium was composed of flat endothelial cells holding several cytoplasmic filaments, lying in blood cysts on a continuous and often laminated basal lamina with many cytoplasmic projections. The results support the hypothesis that cysts of the atrioventricular valves derive from the dilation of blood and lymphatic valvular vessels, do not regress with age, and are mainly the result of mechanical effects. Key words: Atrioventricular valves; blood cysts; cattle; endothelial cells; factor VIII-related antigen; heart; immunohistochemistry; ultrastructure; valvular cysts. Blood cysts of the cardiac atrioventricular (AV) valves, first described in humans in 18443 and subsequently in 1857,15have been frequently reported as incidental necropsy findings in fetuses and infants under 6 months of age, with no pathologic relevance except for giant blood cysts, which may produce valvular stenosis requiring surgical treatment.4. 4, Blood cysts of the AV valves have been described as common congenital lesions that spontaneously tend t o regress with age in dogs, pigs, and but only a few studies have dealt with a significant number of animal^.^^,^^ Here, we report a high frequency of valvular cysts in both young and adult cattle that was recorded during an abattoir survey on bovine cardiac pathology. Besides blood cysts, another less common type of valvular cyst containing a yellow fluid material was observed and referred t o as a serous cyst. The morphology and content of the two types of cysts were investigated by means of histologic, immunohistochemical, electrophoretic, microbiologic, and ultramicroscopic techniques. Material and Methods Hearts from 30,907 cattle slaughtered from June to October 1991 in a private abattoir near Bologna, Italy, were examined to investigate the prevalence of valvular cysts. The animals examined were 5,984 calves (4-5 months of age, Holstein-Friesian males), 15,937 young adults (14-1 5 months of age, Holstein-Friesian and Limousine, 14,024 steers and 1,913 heifers), and 8,986 cows (5-8 years of age, HolsteinFriesian). The hearts were longitudinally sectioned, and the atrioventricular valves were carefully examined for the presence of cysts, which were classified according to their content as blood or serous cysts. When both types of cysts were present in the same heart, the prevailing type only was scored. In a series of 4 12 affected hearts, the valvular cysts were counted and measured and their anatomical location (tricuspid or mitral valve) was recorded. For the histologic examination, representative samples from affected valves (20 blood cysts and 20 serous cysts for each age class) were fixed in 10% buffered formalin and embedded in paraffin. Five-micrometer-thick serial sections were stained with hematoxylin and eosin, Orcein-Van Gieson’s stain for elastic and collagen fibers, Gomori’s stain for reticulin, and 14 Downloaded from vet.sagepub.com by guest on July 25, 2013 Valvular Cysts in Cattle Vet Pathol 33:1, 1996 15 Table 1. Prevalence of cysts of the atrioventricular valves in 30,907 cattle. Prevalence Blood Cysts Serous Cysts Age Class n No. Cysts Calves Young adults Steers Heifers cows 5,984 15,937 14,024 1,913 8,986 687 1,227 1,104 123 1,454 11.48 7.70 7.87 6.43 16.18 408 544 489 55 624 6.82 3.41 3.49 2.88 6.94 279 683 615 68 830 4.66 4.29 4.39 3.55 9.24 Total 30,907 3,368 10.90 1,576 5.10 1,792 5.80 (O4 Perk’ stain for hemosiderh and with the periodic acidachiff (PAS) reaction. For immunohistochemistry, sections from 20 specimens (10 blood cysts and 10 serous cysts) were dewaxed and rehydrated, and endogenous peroxidase was blocked with 3% hydrogen peroxide for 60 minutes at room temperature (RT). The sections were then incubated with Protease XIV (Sigma, St. Louis, MO) 0.05% in Tris buffer for 5 minutes at 37 C. After preincubating for 30 minutes with 1 : 20 normal goat serum (Dakopatts, Copenhagen, Denmark), the sections were allowed to react with the primary antibody, rabbit anti-human Von Willebrand factor (Dakopatts) diluted 1 : 3,000 overnight at 4 C. Biotinylated goat anti-rabbit immunoglobulin (BIO SPA, Milan, Italy; diluted 1 : 100) followed by peroxidase streptavidin (BIO SPA, diluted 1 :250) were used as secondary and tertiary staining reagents, respectively, at RT for 30 minutes. Diaminobenzidine (Sigma) (0.04%, 7 minutes) was used as chromogen, and the sections were then counterstained with Papanicolau hematoxylin. For transmission electron microscopy, valvular cysts samples (three blood cysts and three serous cysts for each age class) were excised from the hearts about 30 minutes after slaughter. Specimens 1-3 mm thick were fixed for 3 hours in 5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, rinsed in the same buffer, and postfixed for 1 hour in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2. After dehydration in a graded sequence of alcohols, the specimens were then embedded in Durcupan AcM resin. Semithin sections were stained for light microscopy with toluidine blue, and ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Philips CMlO transmission electron microscope. Protein concentration of the serous cyst content and of the blood serum from two calves, two steers, one heifer, and two cows was quantified using a commercial assay (Bio Rad, Richmond, CA), and protein analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described. I Gels were stained using Coomassie bnllant blue R-250 and analyzed by densitometric scanning (Model 620 Densitometer, Bio Rad). Molecular weights of single bands were established by comparison with prestained low-molecular-weight markers (Bio Rad). Chloride concentrations of the same samples of blood serum and serous cyst content were determined by a Stat Profile 4 Analyser (Nova Biomedical, Waltham, MA). For microbiologic examination, 0.01 ml from cyst fluid samples (nine blood cysts and nine serous cysts, three samples No. No. % YO for each age class) were cultured on the following solid media: blood agar, Gassner medium, brain-heart infusion, Yoder agar, and Protease-3-agar and on the following fluid media: Yoder broth, SAT medium, and tyoglycolate broth. Cultures were incubated at 37 C in normal atmosphere and in an atmosphere containing 12% CO,. Solid media were incubated for 96 hours and examined at 24-hour intervals. Liquid media were examined every 24 hours, and after 48 hours of incubation 0.0 1-ml samples were transferred to solid media and incubated as indicated above. Results T h e valvular cysts prevalence in the 30,907 exam- ined cattle is listed in Table 1. Macroscopic findings Macroscopically, the cysts were always sessile and roundish or oval and protruding above the atrial surface of the valvular leaflets (Figs. 1, 2). They were 1 mm to 3 cm in diameter. The serous cysts were significantly larger than the blood cysts by analyses of variance (ANOVA; P < 0.001), and the cysts on the mitral valve were larger than those on the tricuspid (P < 0.001). Both types of cysts showed a tendency to enlarge with advancing age (Table 2). Fig. 1. Mitral valve, heart; cow. A large blood cyst (arrow) (diameter = 1.5 cm) and a serous cyst (arrowhead) (diameter = 1.0 cm) are close to the valve base of the two valvular leaflets. Downloaded from vet.sagepub.com by guest on July 25, 2013 Marcato, Benazzi, Bettini, Masi, Della Salda, Sarli, Vecchi, and Poli 16 Vet Pathol 33:1, 1996 Histologic findings Fig. 2. Mitral valve, heart; cow. A large serous cyst (diameter = 1.5 cm) is lying near the valve base. Blood cysts were mostly unilocular and frequently multiple (Table 3) (up to seven on the same valve). In some cases, several blood cysts were grouped together so that the lesion had a multilocular appearance. Serous cysts were unilocular and usually single (Table 3). When the same valve exhibited more than one serous cyst (maximum of three), these were generally located on separate leaflets. The serous cysts were generally close to the base of the valvular leaflet (Fig. 2), but the largest ones showed a tendency to invade the whole surface of the leaflet. Blood cysts were most frequently present on both mitral and tricuspid valves except in cows, where they prevailed on the tricuspid valve; serous cysts were preferably located on the mitral valve in all three age classes (Table 4). A serous cyst was also found on a chordu tendinae of a papillary muscle of the mitral valve in one cow, and a second cyst arose from the endocardium of the left ventricular wall in another cow. Blood and serous cysts were often associated in the same valve (Fig. l), and in some hearts the coexistence of blood and serous cysts was observed in both AV valves. Some microscopic differences were found between blood and serous cysts, independent of the age of the animal. The histologic description therefore applies to all three age classes. Cysts were located in the spongiosa of the valves, which is the looser part of the leaflet. Both types of cysts were internally lined by typical endothelium, lying on an inconspicuous PAS-positive basement membrane. Blood cysts contained intact red blood cells and leukocytes. The wall was mainly composed of several irregular layers of elastic fibers intermingled with some dense collagen and few reticular fibers. Small blood vessels and multilocular spaces of different sizes were present in the wall or close to the cysts, near the valve base. Serial sections revealed dilated blood vessels in direct connection with the blood cyst lumina. No ironpositive macrophages were found near blood cysts. Serous cysts contained a homogenous acidophilic and weakly PAS-positive material, generally devoid of cells except for occasional macrophages containing granules of a yellowish PAS-positive and iron-negative pigment. The wall of the serous cyst was always thinner than that of the blood cyst and lacked dense collagen. No blood vessels were found in the wall. However, a few small endothelium-lined channels were observed, connected with the lumen and directed toward the valve base. No direct connection was observed between cyst lumina and the ventricular cavities. Immunohistochemical findings The endothelium of blood cysts showed a constant positive staining for factor VIII-related antigen that was usually as intense as the staining in the blood vessels of the same section, except that the larger blood cysts showed a weaker and focally interrupted positive staining. The endothelium of serous cysts was always negative for the antigen. Table 2. Diameter (mm) of blood and serous cysts of the atrioventricular valves in 4 12 cattle. Mitral Valve Age Class n Blood Cysts P Mean Range P <0.05 5.0 7.5 10.8 2-15 2-20 1-35 <0.001 <0.01 <0.001 Mean Range Calves* Steers/heiferst Cows§ 79 2.7 140 3.5 193 4.0 1-10 1-15 1-20 Tricuspid Valve Blood Cysts Serous Cysts Serous Cysts NS$ <0.001 Mean Range 2.4 2.9 2.9 * P values for ANOVA of calves versus steers and heifers. *8 t P values for ANOVA of steers and heifers versus cows. NS = not significant. P value for ANOVA of calves versus cows. Downloaded from vet.sagepub.com by guest on July 25, 2013 1-8 1-10 1-8 P <0.01 NS <0.05 Mean Range 3.5 7.0 9.6 2-5 3-15 2-30 P <0.05 NS <0.05 Valvular Cysts in Cattle Vet Pathol 33:1, 1996 17 Table 3. Number of cysts present on the atrioventricular valve leaflets of 412 cattle. Mitral Valve n Age Class Calves Steers and heifers cows 79 140 193 Blood Cysts Tricuspid Valve Serous Cysts Blood Cysts Serous Cysts Mean Range Mean Range Mean Range Mean 2.1 1.5 1.3 1-6 1-4 1-3 1.4 1.2 2-4 1-3 1-3 2.1 1-5 1-7 1.2 1.1 1.2 2.1 1.5 1-7 1.1 Range 1-2 1-2 1-2 mon in calves, especially newborns. Previous reports have stated that these cysts do not usually persist for Blood cysts were internally lined by flat endothelial more than a few months, except in rare cases in which cells having ovoid or convoluted nuclei with irregular they may enlarge and persist for more than 1 year, borders and scarce cytoplasm containing several filaduring which time their content changes into a serous ments, mitochondria, ribosomes, and micropinocyfluid.6,20 totic vesicles (Fig. 3). The cell boundaries showed nuIn one study,2cysts of the AV valves, up to pea-size merous cytoplasmic projections that were fingerlike at and filled with blood or serous-watery fluid, were said the luminal face and rootlike at the abluminal face. to affect up to 75% of calves and to regress during the The endothelial cells lay on a continuous basal lamina life time by resorption and organization phenomena. with frequent aspects of duplication and lamination. The results of our study contrast these widespread The wall of the blood cysts was composed of elastic ideas: 1) blood cysts and serous cysts may be regarded and collagen fibrils and fibroblasts in an amorphous as separate entities; and 2) cysts of the AV valves do matrix containing proteoglycan particles. not regress with age, and their incidence and dimension The endothelial cells lining serous cysts (Fig. 4)were are significantly increased in older animals. similar to those of the blood cysts, although their cyBlood cysts affected both valves, were small, and toplasmic filaments were far more numerous and arcontained fresh blood. Their endothelium was imranged parallel to the basal lamina or encircling the munohistochemically positive for the factor VIII-renucleus. Adjacent endothelial cells were closely conlated antigen, and the wide lamination of the basal nected by extensive overlapping of fingerlike cytolamina was as described in blood vessels end~thelia,~ plasmic projections, and rootlike processes projecting into the underlying tissue were rare. The basal lamina both factors indicative of their blood vessel origin. l 7 was single layered or slightly duplicated, but it never The observed focal discontinuities of the immunoshowed any lamination as seen in blood cysts. The positivity may be the result of the dilation of the larger serous cyst wall was composed of loose connective cysts; as previously noted," the positivity for this antissue rich in proteoglycans, with fibroblasts and scat- tigen becomes weaker as the caliber of vessels increases. tered elastic and rare collagen fibrils. Serous cysts were larger and usually solitary and more Other investigations frequently affected the mitral valve. The lack of enThe results of the biochemical and electrophoretic dothelial reaction to factor VIII-related antigen, the analyses of the serous cyst fluid and blood serum are extensive overlapping of the fingerlike projections of adjacent cells, and the numerous perinuclear filaments shown in Table 5. in the endothelial cells are regarded as distinctive feaMicrobiologic tests were negative for both types of tures of lymph vessel^.^^,'^ cysts. Electrophoresis and biochemical analyses showed an Discussion analogy of components between serous cyst fluid and Hematocysts of the AV valves, up to 1 cm in di- blood serum. Nevertheless, the cyst fluid contained a ameter and sometimes multiple, are considered com- significantly (P = 0.0002) lower concentration of total Ultrastructural findings Table 4. Anatomical location of the valvular cysts of 4 12 cattle. Age Class Calves Steers and heifers cows n 79 140 193 Blood Cysts Serous Cysts Tricuspid Both Mitral Tricuspid 15 (21%) 41 (57%) 16 (22%) 2 (6%) 10 ( 14%) 13 (13%) 37 (33%) 63 (46%) 56 (50%) 36 (26%) 20 (18%) 39 (28%) Downloaded from vet.sagepub.com by guest on July 25, 2013 Both 3 (10%) 5 (7%) 12 (12%) Mitral 26 (84%) 56 (79%) 78 (76%) 18 Marcato, Benazzi, Bettini, Masi, Della Salda, Sarli, Vecchi, and Poli Vet Pathol 33:1, 1996 Fig. 3. Transmission electron micrograph. Mitral valve, heart; steer. The endothelial cells (E) lining the blood cyst lie on a thick and multilayered basal lamina (BL), pierced by rootlike cytoplasmic projections (arrows). Uranyl acetate-lead citrate. Bar = 1 Mm. proteins, with different proportions of albumin and globulin, and a slightly (P = 0.1) higher amount of chloride. These differences indicate that the serous cyst content is similar to lymph.I6x2l Several hypotheses have been formulated to explain the mechanism of valvular blood cyst formation in humans: 1) hematomas, 2) angiomas, 3) ventricular endothelial infoldings of the valve leaflet bulged into the atrium from the pressure gradient during the valve closure and producing valvular diverticula, and 4) ectasia or dilation of blood vessels. Cyst morphology excludes a traumatic or neoplastic genesis. The third hypothesis assumes the existence of connections between the cyst lumina and the ventricles via small endothelium-lined channels,26but these connections have not been observed in this or in studies. Our results support the fourth hypothesis, as recently proposed for canine blood valvular and suggest that bovine blood and serous cysts of the AV valves may originate from the dilation of blood and lymph vessels, respectively. Cysts of the AV valves morphologically equivalent to these bovine serous cysts have been observed in slaughtered pigs and were empirically regarded as distended lymphatic vessels.8JoJs The existence of blood and lymphatic vessels in the AV valves was at one time c o n t r o ~ e r s i abut l ~ ~has ~ been definitely proven in both humans and animals.I2 In cattle, a particularly profuse valvular capillary plexus, which extends to the closing edges of the AV valves, has been described23and may be considered a predisposing factor for the high incidence of valvular cysts in this species. Furthermore, a ventricular subendocardial network of small blood and lymph vessels that drain into branches ascending the papillary muscles and chordae tendinae has been described in the hearts of pigs, dogs, sheep, cattle, and h ~ m a n s . The ~ , ~two ~ atypical serous cysts observed in a chorda tendinae and in the left ventricular wall of two cows in this study were located along the course of lymph vessels. The higher frequency of larger valvular cysts in cows than in young cattle contrasts with the general opinion Downloaded from vet.sagepub.com by guest on July 25, 2013 Valvular Cysts in Cattle Vet Pathol 33:1, 1996 19 Fig. 4. Transmission electron micrograph. Mitral valve, heart; steer. There is extensive overlapping (arrow) of the cytoplasmic projections of two adjacent endothelial cell (E) in a serous cyst. Uranyl acetate-lead citrate. Bar = 1 pm. that valvular cysts are congenital lesions that regress during the first year of life. Our observations support the hypothesis that valvular cysts persist and enlarge during the entire life time and that they may also be acquired later in life. Likewise, low-degree congenital ectasias of vessels might become grossly observable only after age-related enlargement. Etiologic factors leading to blood cyst formation are unknown. It has been supposed that mechanical effects, such as an increase in tension, friction, and impact, can trigger the enlargement of valvular blood vessels in a sort of valvular telangiectasis with subsequent cyst formation.24 Sex does not appear to play any role in the development of valvular cysts. The fact that all the affected calves were male and all the adults were cows was only a sampling artifact. The group of young adults, formed by male and female cattle, showed a similar incidence of cysts in both sexes. In addition to the progressive enlargement of the valvular cysts, advancing age seems to correlate with a decrease in the number of blood cysts and an increase Table 5. Electrophoretic and biochemical analysis of the fluid contained in the serous cysts and of the homologous blood serum. Sample Total protein Wdl) Mean Serous cyst content Blood serum Range 2.9 2.2-4.3 6.0 5.1-7.0 P* 0.0002 * t-test for paired samples. Albumin Wdl) Mean Range 1.35 0.85-2.9 1.77 1.09-2.38 0.08 Immunoglobulin (g/dl) Mean Range 0.06 0.04-0.1 0.36 0.25-0.59 0.0009 Downloaded from vet.sagepub.com by guest on July 25, 2013 Hemoglobin (g/W Mean Range 0.007 0.36 0.02 0-0.04 0.13-0.8 Chloride (mEq/liter) Mean Range 123.8 120-129 120.8 116-124 0.1 20 Marcato, Benazzi, Bettini, Masi, Della Salda, Sarli, Vecchi, and Poli - Specific bovine predisposition: - profuse valvular capillary plexus - ossification of the anulus fibrosus Fig. 5. Suggested pathogenetic mechanism for the valvular cyst formation in cattle. in the number of serous cysts. A change from blood to serous contentZodoes not seem likely, however, because blood and serous cysts have a different predilection for mitral and tricuspid valves and a different anatomical structure. Moreover, in this large sample of animals no form of transition between blood and serous cysts was observed. The predilection of the serous cysts for the mitral valve could be related t o a lower capacity of the lymphatics, which have thinner walls than hematic vessels, to withstand the dilation consequent from the systolic pressure gradient, which is stronger in the left ventricle than in the right one. This explanation may also suffice for the larger dimension of the cysts located on the mitral valve. At the atrial surface of the tricuspid and mitral valves, there are lymph capillaries that extend from the free margins of the cusps t o the anulusfibrosus of each valve, where they drain into a channel passing through the myocardium and joining the subepicardial duct of the atrioventricular S U ~ C U S . ~ The bovine physiologic age-related ossification of the anulus Jibrosus may hinder the lymph outflow through the transmyocardial channels and facilitate the dilation of the valvular lymph vessels, which may thus explain the high frequency and large dimensions of serous cysts in adult cattle. Other age-related predisposing factors (Fig. 5 ) would be the progressive obstruction and diminution of the valvular blood and lymph vessels9Jz and the progressive loosening of the spongiosa stratum of the AV valves.' References 1 Boyd TAB: Blood cysts on the heart valves of infants. Am J Pathol 25757-759, 1949 Vet Pathol 33:1, 1996 2 Drommer W: Kreislauforgane. In: Pathologie der Haustiere, ed. 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Cardiovasc Res 5: 194-200, 197l 24 Takeda T, Makita T, Nakamura N, Kimizuka G: Mor- Downloaded from vet.sagepub.com by guest on July 25, 2013 Vet Path01 331, 1996 Valvular Cysts in Cattle phologic aspects and morphogenesis of blood cysts on canine cardiac valves. Vet Path01 28: 16-2 1, 199 1 25 Van Vleet JF, Ferrans VJ: Cardiovascular system. In: Special Veterinary Pathology, ed. Thomson RG, p. 3 15. Decker, Toronto, OW, Canada, 1988 21 26 Zimmerman KG, Paplanus SH, Dong S , Nagle RB: Congenital blood cysts of the heart valves. Hum Path01 14: 699-703, 1983 Request reprints from Prof. P. S. Marcato, Istituto di Patologia Generale e Anatomia Patologica Veterinaria, Via Tolara di Sopra 50,I-40064, Ozzano Emilia, Bologna (Italy). Downloaded from vet.sagepub.com by guest on July 25, 2013