Reassessment of spiny dogfish Squalus acanthias age and growth using vertebrae and dorsal-fin spines.

Male and female spiny dogfish Squalus acanthias were collected in the western North Atlantic Ocean in the Gulf of Maine between July 2006 and June 2009. Squalus acanthias ranged from 25 to 102 cm stretch total length and were caught during all months of the year except January. Age estimates derived from banding patterns visible in both the vertebrae and second dorsal-fin spines were compared. Vertebral growth increments were visualized using a modified histological staining technique, which was verified as appropriate for obtaining age estimates. Marginal increment analysis of vertebrae verified the increment periodicity, suggesting annual band deposition. Based on increased precision and accuracy of age estimates, as well as more biologically realistic parameters generated in growth models, the current study found that vertebrae provided a more reliable and accurate means of estimating age in S. acanthias than the second dorsal-fin spine. Age estimates obtained from vertebrae ranged from <1 year-old to 17 years for male and 24 years for female S. acanthias. The two-parameter von Bertalanffy growth model fit to vertebrae-derived age estimates produced parameters of L∞ = 94·23 cm and k = 0·11 for males and L∞ = 100·76 cm and k = 0·12 for females. While these growth parameters differed from those previously reported for S. acanthias in the western North Atlantic Ocean, the causes of such differences were beyond the scope of the current study and remain to be determined.

Short communication: Rapid antibiotic screening tests detect antibiotic residues in powdered milk products.

Rapid antibiotic screening tests are widely used in the dairy industry to monitor milk for the presence of antibiotic residues above regulated levels. Given the persistent concern over contamination of milk products with antibiotic residues, we investigated the utility of IDEXX Snap test devices (IDEXX Laboratories Inc., Westbrook, ME) as tools for detecting antibiotic residues in powdered milk products. Five powdered milk products were reconstituted according to manufacturer specification with distilled water: Carnation (Nestlé USA Inc., Solon, OH), Nido youth and Nido adult (Nestlé Mexico Inc., Mexico City, Mexico), ELK (Campina, Eindhoven, the Netherlands), and Regilait (Saint-Martin-Belle-Roche, France). Positive samples were generated by spiking reconstituted milk with penicillin G, cephapirin, or tetracycline to either the European Union-regulated maximum residue limit or the FDA-regulated safe/tolerance level, whichever was lower. Control, unspiked negative milk samples and positive samples were tested with appropriate IDEXX Snap test kits (penicillin G and cephapirin with New Beta-Lactam, tetracycline with New Tetracycline). All samples yielded definitive results consistent with expectations, and there were no instances of false-positive or false-negative readings. These results suggest that both the New Beta-Lactam and New Tetracycline IDEXX Snap test kits effectively detect antibiotic residues in commercially available powdered milk samples and are useful tools for monitoring antibiotic residues in reconstituted powdered milk products.

Age and size at sexual maturity of the smooth skate Malacoraja senta from the western Gulf of Maine.

Age and size at sexual maturity was determined for 185 male and 96 female smooth skates Malacoraja senta (ranging in size from 370 to 680 mm total length L(T)), collected from the western Gulf of Maine. Maturity ogives for males, based on clasper length, testis mass and the proportion of mature spermatocysts in the testes, suggest that 50% maturity occurs between 9 and 10 years and 560 mm L(T). Maturity ogives for females, based on ovary mass, shell-gland mass and maximum follicle size, suggest that 50% maturity occurs at age 9 years and 540 mm L(T).

Size and age estimates at sexual maturity for the little skate Leucoraja erinacea from the western Gulf of Maine, U.S.A.

Size and age estimates at sexual maturity were determined for 162 male and 273 female little skates Leucoraja erinacea collected from the western Gulf of Maine. Maturity ogives suggest that 50% maturity in females occurs at age 9.5 years and 480 mm total length (LT), whereas 50% maturity in males occurs at a slightly younger age of 7.7 years and smaller size of 460 mm LT. Age estimates were made from 389 L. erinacea ranging in size from 93 to 570 mm LT. The index of average per cent error and age-bias plots indicated that the ageing methods were precise and non-biased. Additionally, annual periodicity of band formation was validated with oxytetracycline in eight individuals (three males and five females) ranging in age from 3 to 12 years. In conclusion, results from this study indicate that L. erinacea exhibits characteristics that make other elasmobranch populations highly susceptible to overexploitation.

Angiogenic proteins in the lungs of children after cavopulmonary anastomosis.

OBJECTIVE: Pulmonary arteriovenous malformations may cause progressive cyanosis after cavopulmonary anastomosis and may develop as a result of abnormal angiogenesis. We used immunohistochemistry to determine whether angiogenic proteins are increased in the lungs of children after cavopulmonary anastomosis. METHODS: Lung specimens were obtained from 13 children after cavopulmonary anastomosis and from 6 control subjects. Specimens were stained with antibodies against vascular endothelial growth factor and its receptor (flk-1/KDR), basic fibroblast growth factor, alpha-smooth muscle actin, CD31, collagen IV, fibronectin, and proliferating cell nuclear antigen. Staining was graded on a scale of 0 to 3. Vessels positive for proliferating cell nuclear antigen were counted in 10 fields per specimen, and the results were averaged. RESULTS: After cavopulmonary anastomosis, patients demonstrated increased staining for vascular endothelial growth factor (P =.03) and its receptor (P =.03) and decreased staining for CD31 (P =.004). Proliferating cell nuclear antigen staining in patients was equivalent to that for control subjects (P =.9). CONCLUSIONS: Lung biopsy specimens from children after cavopulmonary anastomosis demonstrate increased expression of vascular endothelial growth factor and its receptor. These data confirm earlier findings that blood vessels forming after cavopulmonary anastomosis may have reduced intercellular junctions (decreased CD31 staining). Despite the increased numbers of pulmonary vessels that are present in these patients, these vessels are not highly proliferative (proliferating cell nuclear antigen staining equivalent to that of control subjects). These results suggest that vascular endothelial growth factor may be a mediator of angiogenesis in the lungs of children after cavopulmonary anastomosis; however, other factors, such as vascular dilation and remodeling, may also be important.

Pulmonary microvessel density is a marker of angiogenesis in children after cavopulmonary anastomosis.

OBJECTIVE: Pulmonary arteriovenous malformations cause progressive cyanosis in children after cavopulmonary anastomosis and may be due to abnormal angiogenesis. We determined the microvessel density, a marker of angiogenesis, in the lungs of children after cavopulmonary anastomosis. METHODS: Lung biopsy specimens were obtained from 8 children after cavopulmonary anastomosis and from 4 control patients. Three of the 8 children undergoing cavopulmonary anastomosis had clinical and angiographic evidence of pulmonary arteriovenous malformations, whereas the other 5 were free of symptoms. Routine histologic and immunohistologic stains were performed with a primary antibody to von Willebrand factor. Microvessel staining for von Willebrand factor was determined for 10 fields (200x) per patient. RESULTS: Patients with and without pulmonary arteriovenous malformations after cavopulmonary anastomosis demonstrated significantly increased microvessel density compared with control subjects (32.7 +/- 2.8 vs 9.3 +/- 4.6, P =.02, and 31.5 +/- 15.7 vs 9.3 +/- 4.6, P =.01, respectively). There was no difference in microvessel density in children with and without clinically apparent pulmonary arteriovenous malformations after cavopulmonary anastomosis (P =.9). The children with pulmonary arteriovenous malformations had numerous greatly dilated vessels that were absent in the asymptomatic children after cavopulmonary anastomosis. CONCLUSIONS: After cavopulmonary anastomosis, pulmonary microvessel density is increased even in the absence of clinically apparent pulmonary arteriovenous malformations, supporting the presence of a constant angiogenic stimulus. Children with clinically apparent pulmonary arteriovenous malformations possess large numbers of greatly dilated pulmonary microvessels, which are absent in asymptomatic children after cavopulmonary anastomosis. These results suggest that the transition to clinically apparent pulmonary arteriovenous malformations may be due to mechanisms that lead to vessel dilation and remodeling.

Procollagen synthesis by fresh and cryopreserved rat pulmonary valve grafts.

OBJECTIVE: Allograft valves are frequently used in the repair of congenital cardiac anomalies. The failure rate may differ depending on the type of allograft used. Previous studies have shown that rat aortic valve grafts exhibit synthesis of procollagen, suggesting a capacity for repair and regeneration after implantation. No studies of pulmonary valve grafts in the heterotopic rat implant model have thus far been reported. This study was designed to investigate whether pulmonary valve grafts maintain in vivo viability, as demonstrated by procollagen synthesis, and whether cryopreservation, histocompatibility, or both affect this property. METHODS: Cryopreserved and fresh rat pulmonary valves were implanted into the abdominal aorta of syngeneic and allogeneic recipients. The grafts and native valves were excised 3 to 21 days after implantation. Valves were sectioned and immunohistochemically stained for procollagen. Computerized morphometry was used to calculate changes in intima, media, and adventitia as a percentage of cross-sectional area of the graft. Procollagen content was graded by semiquantitative methods. RESULTS: Pulmonary valve grafts had significantly greater collagen density in the intima and adventitia compared with native aortic and pulmonary tissues, but collagen density in the media was similar in all groups. The grafts demonstrated appreciably greater procollagen than the corresponding native valves. These findings were consistent in all grafts (ie, both fresh and cryopreserved, both syngeneic and allogeneic), irrespective of duration of implantation. CONCLUSIONS: Procollagen synthesis occurs in pulmonary valve grafts early after implantation, indicating viability of these tissues. This model of pulmonary valve implantation may have wide applicability to questions of allograft biology.

Vascular endothelial growth factor and basic fibroblast growth factor in children with cyanotic congenital heart disease.

OBJECTIVE: Vascular endothelial growth factor and basic fibroblast growth factor are potent stimulators of angiogenesis. Children with cyanotic congenital heart disease often experience the development of widespread formation of collateral blood vessels, which may represent a form of abnormal angiogenesis. We undertook the present study to determine whether children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor and basic fibroblast growth factor. METHODS: Serum was obtained from 22 children with cyanotic congenital heart disease and 19 children with acyanotic heart disease during cardiac catheterization. Samples were taken from the superior vena cava, inferior vena cava, and a systemic artery. Vascular endothelial growth factor and basic fibroblast growth factor levels were measured in the serum from each of these sites by enzyme-linked immunosorbent assay. RESULTS: Vascular endothelial growth factor was significantly elevated in the superior vena cava (P =.04) and systemic artery (P =.02) but not in the inferior vena cava (P =.2) of children with cyanotic congenital heart disease compared to children with acyanotic heart disease. The mean vascular endothelial growth factor level, determined by averaging the means of all 3 sites, was also significantly elevated (P =.03). Basic fibroblast growth factor was only significantly elevated in the systemic artery (P =.02). CONCLUSION: Children with cyanotic congenital heart disease have elevated systemic levels of vascular endothelial growth factor. These findings suggest that the widespread formation of collateral vessels in these children may be mediated by vascular endothelial growth factor.

A detailed histologic analysis of pulmonary arteriovenous malformations in children with cyanotic congenital heart disease.

INTRODUCTION: Pulmonary arteriovenous malformations are a common cause of progressive cyanosis in children after cavopulmonary anastomoses. We analyzed the pulmonary histologic characteristics from children in whom pulmonary arteriovenous malformations developed after procedures that resulted in pulmonary arterial blood flow devoid of hepatic venous effluent. METHODS: We performed routine histologic studies, immunohistochemical staining, and electron microscopic analysis of peripheral lung biopsy specimens from 2 children with angiographically proven pulmonary arteriovenous malformations. Microvessel density was determined with a computer-assisted, morphometric analysis system. RESULTS: Histologic examination demonstrated large, dilated blood vessels ("lakes") and clustered, smaller vessels ("chains") in the pulmonary parenchyma. Microvessel density was significantly greater in these patients than in age-matched controls (P =.01). Immunohistochemistry demonstrated uniform staining for type IV collagen and alpha-smooth muscle actin, weak staining for the endothelial marker CD31 (cluster of differentiation, PECAM-1), and negative staining for proliferating cell nuclear antigen. Electron microscopy revealed endothelial irregularity, a disorganized basement membrane, and increased numbers of collagen and actin filaments beneath the endothelium. CONCLUSIONS: This study represents an attempt to characterize the histologic features of pulmonary arteriovenous malformations in children with congenital heart disease who have pulmonary arterial blood flow devoid of hepatic venous effluent. The histologic correlate of this condition appears to be greatly increased numbers of thin-walled vessels. Immunohistochemistry suggests that the rate of cellular proliferation is not increased in these lesions. The development of these techniques may provide a standardized histologic approach for this condition and aid in understanding its etiology.

Effect of cryopreservation and histocompatibility on type I procollagen gene expression in aortic valve grafts.

BACKGROUND: Allograft valves are excellent substitutes for diseased or absent valves but undergo primary tissue degeneration. Fibroblast viability may determine resistance to valve deterioration. This study evaluated gene expression for procollagen by valve grafts and studied the effects of cryopreservation and histocompatibility on this property. METHODS AND RESULTS: Fresh and cryopreserved rat aortic valves were implanted heterotopically into syngeneic or allogeneic recipients. Nonviable, cryothermally injured valves were used as negative controls. The grafts and native aortic roots were excised 3 days after implantation. Northern hybridization with a human procollagen alpha 1 (I) complementary DNA probe was used to assess the expression of type I procollagen mRNA. The content of procollagen mRNA relative to 18S ribosomal RNA was evaluated by means of scanning densitometry. In situ hybridization was used to locate the areas of procollagen mRNA expression in the grafts. Both fresh and cryopreserved grafts exhibited greater expression than the native valve. This increase in expression was observed in both syngeneic and allogeneic grafts, but not in the negative control group. In situ hybridization showed a strong signal for procollagen in the aortic wall and a weak signal in the leaflet and myocardium in the viable grafts and in native tissues. CONCLUSIONS: Regardless of preservation or allogenicity, fibroblast viability in aortic valve grafts persists after implantation. Increased gene expression for procollagen suggests a capacity for repair and regeneration.

Collagen synthesis is upregulated in mitral valves subjected to altered stress.

Mitral regurgitation (MR) and abnormal ventricular wall motion (AVWM) are two cardiac conditions that may increase mitral valve (MV) stresses. Theoretically, increased stress could induce damaging MV tissue alterations. These alterations may impair the preferred option of repair, and mandate replacement. It is hypothesized that MV collagen synthesis is upregulated in response to MR and AVWM. To test this hypothesis in a pilot study, an ischemic sheep model (n = 8) was employed. Four sheep underwent selective coronary artery ligation to infarct a papillary muscle, which resulted in MR. Two other sheep underwent similar coronary ligation to create AVWM. As controls, two sheep underwent sham surgery (no ligation). Sheep were killed 4 and 8 weeks post operatively and their MVs were sectioned. Sections were stained with an antibody (SP1.D8, University of Iowa) to procollagen I (precursor to collagen I). The percent area of procollagen stain present present was measured by image analysis (Optimas Corporation) and used as an indicator of collagen synthesis. Procollagen results indicated that MV collagen synthesis was upregulated by factor of 1.8 in both the MR and AVWM groups versus controls. In addition, results showed greater upregulation in anterior leaflets compared with posterior leaflets in both infarct groups. These results indicate that MV collagen synthesis is upregulated in response to MR and AVWM.

Procollagen production in fresh and cryopreserved aortic valve grafts.

Long-term durability of aortic valve allografts may be enhanced by cellular capacities for regeneration and repair. To evaluate aortic valve graft production of an important structural protein, rat aortic roots were implanted heterotopically into the abdominal aorta of recipient rats. Grafts were either syngeneic or strongly allogeneic, were implanted either fresh or after cryopreservation, and were left in place 2 to 21 days after implantation. A total of 80 aortic valve grafts and the corresponding native aortic valves were examined. The grafts were retrieved and immunocytochemically stained for the presence of procollagen, a precursor to collagen. Regardless of histocompatibility or preservation, grafts exhibited consistent procollagen presence that equaled or exceeded that seen in the corresponding native valves. Positive procollagen staining was predominantly in the aortic wall. The most prominent staining was near the hinge point of the valve leaflets, with no staining in the free portion of the leaflets. Staining with alpha-actin demonstrated vascular smooth muscle in sites remote from the areas positive for procollagen, which suggests that vascular smooth muscle was not responsible for the procollagen production. These findings indicate that cryopreservation is compatible with persistent fibroblast viability and in vivo protein synthesis by both syngeneic and allogeneic aortic valve grafts.

Effects of preservation techniques on in vivo expression of adhesion molecules by aortic valve allografts.

Methods of sterilization and preservation of aortic valve allografts influence graft longevity. The effect of storage techniques on valve durability may be mediated by alterations in the immunologic properties of the allograft, which are reflected by expression of leukocyte adhesion molecules. Rat aortic valve grafts were transplanted in the fresh state, after cryopreservation (-196 degrees C), or after storage at 4 degrees C for 1 to 21 days. Syngeneic and strongly allogeneic valves were transplanted for 4 hours to 21 days and were retrieved for immunohistochemical staining for expression of leukocyte adhesion molecules. Unimplanted valves and transplanted syngeneic valves, regardless of storage methods, exhibited little or no expression of leukocyte adhesion molecules. Fresh allogeneic valves expressed all molecules, indicating up-regulation, at all time intervals studied. Cryopreserved allogeneic valves demonstrated no leukocyte adhesion molecules at 4 hours or 2 days and weak reactivity at 10 and 21 days. Allogeneic valves stored at 4 degrees C, regardless of the duration of storage, demonstrated weak expression of all molecules at 10 days and strong expression at 21 days. Expression of leukocyte adhesion molecules requires an allogeneic environment and may precede immune-mediated injury. Reduced expression of leukocyte adhesion molecules resulting from storage may predict a diminished immunologic response. Cryopreservation (-196 degrees C) causes the greatest delay and diminution of expression of leukocyte adhesion molecules.

Effect of cryopreservation on the presence of endothelial cells on human valve allografts.

Cryopreserved human allograft valves are useful in a variety of cardiac operations. The presence or absence of endothelial cells on allografts may be important in determining immunogenicity and ultimate graft longevity. The purpose of this study was to determine whether endothelial cells are present on cryopreserved human allografts. Portions of cryopreserved allografts (35 valve leaflets, 96 pieces of arterial wall) not used at operation were studied. For comparison, untreated tissues (44 valve leaflets, 46 pieces of arterial wall) were obtained from structurally normal hearts and lungs removed or inserted at the time of transplantation and from pathologic tissues obtained during operations for congenital heart defects. A monolayer of cells from the luminal surface of each specimen was harvested by means of a Hautchen preparation. The monolayer was stained with fluorescein isothiocyanate-labeled Ulex europaeus I, a lectin with strong affinity for human endothelium. Positive staining with fluorescein was considered to be evidence for the presence of human endothelium. Endothelial cells were observed on 21 of 131 (16%) cryopreserved allograft specimens and on 70 of 90 (78%) untreated tissues (p < 0.001). These results show that cryopreservation typically results in the loss of endothelium from aortic and pulmonary valve allografts. These findings may have important implications for the immunologic response of the host to allograft implantation.

Endothelial cell replication in an in vivo model of aortic allografts.

Previous studies of aortic valve allograft viability have used in vitro assessments that may not reflect in vivo properties. This study evaluated in vivo endothelial cell replication in experimental valved aortic grafts and examined the consequences of histoincompatibility and cryopreservation. Valved aortic conduits were heterotopically transplanted into syngeneic or allogeneic rats. Tritiated thymidine was administered to graft recipients and control rats. After 72 hours, monolayers from the native aortas and the aortic portion of the grafts were prepared for autoradiography, with six or more silver grains per nucleus considered evidence of replication. Percentages of replicating cells in native aortas ranged from 0.3% to 2.3% (p = not significant). Percentages of replicating cells in the fresh isografts (12.4%) and allografts (12.2%) were not significantly different from each other, although each was significantly greater than the percentage in its native aorta (p < 0.04). Cryopreserved allografts and isografts displayed a few endothelial cells, none of which was replicating. Immunologic differences do not affect endothelial cell replication in this early period after fresh graft transplantation. Cryopreservation, however, results in the absence of replicating endothelium.