Comparative analysis between ventricular and sinoatrial node vascularization in cats

Biasi C., Borelli V., Benedicto H.G., Pereira M.R., Favaron P.O. & Bombonato P.P. 2012. [Comparative analysis between ventricular and sinoatrial node vascularization in cats.] Analise comparativa entre a vascularizacao ventricular e do no sinoatrial em gatos. Pesquisa Veterinaria Brasileira 31(1): 78-82. Setor de Anatomia dos Animais Domesticos e Silvestres, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitaria, Sao Paulo, SP 05508-000, Brazil. E-mail: caiobiasi@usp.br The possible existence of interdependence in the blood nutrition of both atrial and ventricular territories has been a subject of concern to cardiologists, mainly related to vascularization of the sinoatrial node and its dependence on just one coronary artery or both, and its relation with the predominance of these vessels in the ventricular vascularization. Therefore, this research aimed evaluated the relation of blood irrigation of the sinoatrial node in relation to the coronary artery predominance in the ventricle vascularization. In doing so, we analyzed 30 hearts of cats without pedigree, males and females, adults of several ages. They were not carrying any heart problems. The hearts were injected by the thoracic aorta with Neoprene Latex 450, stained with red pigment, and then they were dissected. It was found that when there was a prevalence of ventricular vascularization of the left type (63.34%) the sinoatrial node irrigation was predominantly in the dependency of the Ramus proximalis atrii dextri (78.9%) or with less frequency by Ramus proximalis atrii sinister (21.1%). In the ventricular vascularization of the balanced type (33.34%), the pacemaker irrigation was in dependence more often of Ramus proximalis atrii dextri (80%) or with less frequency the nutrition of the sinoatrial node occurred by Ramus proximalis atril sinister (20%). In a single-case, we observed the ventricular vascularization of the right type (3.34%), the pacemaker nutrition was in an exclusive dependence of the Ramus intermedius atril dextri. These results suggest in this species there is no relationship between both the sinoatrial node irrigation and the type of ventricular vascularization, regardless of gender.

Prenatal retinoic acid improves lung vascularization and VEGF expression in CDH rat

OBJECTIVE: We sought to investigate the effects of antenatal retinoic acid on the pulmonary vasculature and vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) expression in a nitrofen-induced congenital diaphragmatic hernia (CDH) model. STUDY DESIGN: Rat fetuses were exposed to nitrofen at gestational day 9.5 and/or all-trans retinoic acid (ATRA) at gestational days 18.5-20.5. We assessed lung growth, airway, and vascular morphometry. VEGF, VEGFR1, and VEGFR2 expression was analyzed by Western blotting and immunohistochemistry. Continuous data were analyzed by analysis of variance and Kruskal-Wallis test. RESULTS: CDH decreased lung to body weight ratio, increased mean linear intercept and mean transection length/airspace, and decreased mean airspace cord length. ATRA did not affect lung growth or morphometry. CDH increased proportional medial wall thickness of arterioles while ATRA reduced it. ATRA recovered expression of VEGF and receptors, which were reduced in CDH. CONCLUSION: Retinoic acid and VEGF may provide pathways for preventing pulmonary hypertension in CDH.

Antiproliferative Effects of Fluoxetine on Colon Cancer Cells and in a Colonic Carcinogen Mouse Model

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

Kidney of Minke Whale (Baleanoptera acutorostrata): Architecture and structure

Sarmento C. A. P., Ferreira A. O., Rodrigues E. A. F., Lesnau G. G., Rici R. E. G., Abreu D. K., Biasi C. & Miglino M. A. 2012. [Kidney of Minke Whale (Baleanoptera acutorostrata): Architecture and structure.] Rins de Baleia Minke (Baleanoptera acutorostrata): arquitetura e estrutura. Pesquisa Veterinaria Brasileira 32(8): 807-811. Departamento de Cirurgia, Setor de Anatomia dos Animais Domesticos e Silvestres, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Sao Paulo, SP 05508-270, Brazil. E-mail: sarmento@usp.br Among marine mammals, whale is one of the most attention-arousing animals, especially concerning its urinary tract. This system follows the pattern of mammals with regard to its constitution, however, it differs in renal morphology and number of lobes, which, in turn, form complete reniculi, agglutinated in hundreds. This structure is supported by fibrous connective tissue, but highly capable of maintaining electrolyte balance. Six pairs of kidneys of Minke whale (Balaenoptera acutorostrata), collected in 1982, in Cabedelo, Paraiba, Brazil, in the last fishing allowed, were dissected. These kidneys were preserved in 10% formaldehyde and they presented a very large histologic layer of collagen surrounding the medullary wall. The urinary collecting duct form papillary glasses, that reach a single collecting center which discharges in the ureter. It was found that the kidney of Minke whale has a lobe characteristic, with, on average, 700 reniculi; each reniculus has anatomical and functional characteristics of a unipyramidal kidney, with an inner layer (medulla), and an outer layer (cortex), and independent irrigation, with formation of individually arcuate arteries, as observed in unipyramidal terrestrial mammals. However, the set gathering all these reniculi constitutes, in the end, a multilobular and polipyramidal kidney, contrary to the morphology of most terrestrial mammals. It was not possible to distinguish the renicular cortex structures of the Minke whale in the level of light microscopy. Through scanning electron microscopy, it was possible to visualize a cortical layer located between two fibrous capsules. This joint, in turn, consists of connective tissue, which, along with a layer of collagen and elastic fibers, separates the cortex from the medulla; the kidney glomeruli were visualized, completely taken by the glomerular vessels and arranged into several layers. One notices that the glomerular cavity is almost a virtual space into which the glomerular filtrate is drained, and it does not present a globular shape. Vascularization is increased in the medullary region. The difference between the kidneys of terrestrial and marine mammals consists in the arrangement of morphological components, favoring the organ's physiology.

Microvascularization on Collared Peccary Placenta: a Microvascular Cast Atudy in Late Pregnancy

The microvascularization of the collared peccary (Tayassu tajacu) placenta was studied by vascular casts and immunolocalization of alpha-smooth muscle actin and vimentin, to identify the three-dimensional organization and vascular flow interrelation in the microvasculature between the maternal and fetal compartments of the placentae. The immunolocalization of vimentin in the vascular endothelium and in the smooth muscle cells of blood vessels showed indented capillaries along the uterine epithelium and the trophoblast at the sides of complementary maternal and fetal microfolds, or rugae. This confers the three-dimensional structure observed in vascular casts. On the maternal side, casts demonstrated uterine folds coated by with primary and secondary ridges, and by areolae dispersed between these ridges. The arteriole runs through the center/middle of ridges, branching at the top into a microvascular network wall in a basket-like fashion. At the base of these baskets venules were formed. On the fetal side, arterioles branched centrally in the fetal rugae into a capillary network in a bulbous form, complementary to the opposite maternal depressions forming the baskets. At the base of the bulbous protrusions, the fetal venules arise. The blood vessel orientation in the materno-fetal interface of the placentae of collared peccaries suggests a blood flow pattern of the type countercurrent to crosscurrent. The same pattern has been reported in domestic swine demonstrating that, even after 38 million years, the Tayassuidae and Suidae families exhibit similar placental morphology, which is here characterized at the microvascular level.

Morphology and Involution of the Yolk Sac during Early Gestation Bovine (Bos indicus)

Background: The bovine yolk sac derives from visceral endoderm and its development occurs between days 18-23 of gestation. The study of this membrane is important for comparative data and has already been performed in rodents, sheep and in cattle, especially Bos taunts. In species Bos indicus the yolk sac has not quite been studied and is believed that there are morphological differences between these species. The yolk sac undergoes a process of involution and degeneration during embryonic development and none vestige of it is found in late gestation. The period in which occurs the involution of the yolk sac coincides with the period of increased pregnancy loss in cattle, and changes in the morphology of this membrane may indicate the reasons for such high loss rates. Thus, considering that the yolk sac is important for embryonic circulation and metabolic transmission, besides participating actively in the process of cattle placentation, this study aimed characterize morphologically the involution of the bovine yolk sac. Materials, Methods & Results: The early gestational period was determined between days 20 and 70 post-insemination (p.i), according to the exterior characteristics of embryo/fetus. For macroscopic analyzes the uterus was dissected to expose the fetal membranes and subsequently the embryo/fetus was photographed. The samples were fixed for light microscopy and transmission electron microscopy. The yolk sac that emerges from the ventral part of the embryo was prominent and composed by a central part with two thin peripheral projections of different lengths. The bovine yolk sac with about 9 cm on day 25 p. i. of pregnancy permanently decreased its total length during this study. Histologically, the yolk sac is composed of three cell layers: the mesothelium, the mesenchyme and the endoderm. In mesenchyme are found blood islets. In the endoderm are formed cells invaginations toward the mesenchyme originating small canaliculi. The ultrastructure of yolk cells presented many mitochondria, rough endoplasmic reticulum, vesicles, euchromatin and the presence of two nucleoli, Discussion: The real first blood circulation in the bovine is attached with the development of yolk sac, differently from other membranes, such as the corium, that does not present evidence of vascularization by the age of 20-30 days. The erythroblasts found in the yolk sac are related to vasculogenesis and the process of differentiation of blood cells during the erythropoiesis. It could be observed on the histology of the yolk sac, in embryos of 30-50 days old, the presence of canaliculi and small folds of the epithelium. The canaliculi collapse is associated with the degeneration of the endoderm wall of the yolk sac. The organelles present in the endoderm cells of the yolk sac are associated with the function of protein metabolism and in the exchange of substances between the mesenchyme and the mesothelium, For these findings, could be observed that the yolk sac epithelium is found active until the 50th day of gestation, and thereafter regresses. However, remnants of this membrane may be present until the 70th day, These features may represent a presence of an active chorionvitelline placenta in this period responsible for the maintenance of pregnancy whereas the chorioallantoic placenta is not definitively established.

Morfologia dos vasos da base do encéfalo do quati (Nasua nasua)

Estudou-se a morfologia do encéfalo de Nasua nasua - quati, buscando comparar estes achados com outras espécies já descritos. Foram utilizados cinco encéfalos de quatis, provenientes do Criatório Científico (Cecrimpas), Unifeob. Os animais foram eutanásiados de acordo com a legislação (Cobea). Canulou-se a artéria carótida comum e a veia jugular externa sentido cranial, injetou-se solução de látex/bário corado de vermelho na artéria carótida, e solução de látex corado de azul na veia jugular. Em seguida os animais fixados em solução de formaldeído a 10%. O encéfalo tem sua nutrição dependente de quatro artérias, as artérias carótidas internas e as artérias vertebrais direitas e esquerdas. Esses vasos compuseram o circuito basilar e carotídeo que se anastomosam através das artérias cerebrais caudais. Correm na base do encéfalo sob a meninge pia mater.

Fetal Pulmonary Response After Fetoscopic Tracheal Occlusion for Severe Isolated Congenital Diaphragmatic Hernia

OBJECTIVE: To estimate the response in lung growth and vascularity after fetal endoscopic tracheal occlusion for severe congenital diaphragmatic hernia in the prediction of neonatal survival. METHODS: Between January 2006 and December 2010, fetal lung parameters (observed-to-expected lung-to-head ratio; observed-to-expected lung volume; and contralateral lung vascularization index) were evaluated before fetal tracheal occlusion and were evaluated longitudinally every 2 weeks in 72 fetuses with severe isolated congenital diaphragmatic hernia. Thirty-five fetuses underwent fetal endoscopic tracheal occlusion and 37 cases did not. RESULTS: Survival rate was significantly higher in the fetal endoscopic tracheal occlusion group (54.3%) than in the no fetal endoscopic tracheal occlusion group (5.4%, P<.01). Fetal endoscopic tracheal occlusion resulted in a significant improvement in fetal lung size and pulmonary vascularity when compared with fetuses that did not go to the fetal intervention (increase of the observed-to-expected lung-to-head ratio, observed-to-expected total lung volume, and contralateral pulmonary vascularization index 56.2% compared with 0.3%, 37.9% compared with 0.1%, and 98.6% compared with 0.0%, respectively; P<.01). Receiver operating characteristic curves indicated that the observed-to-expected total fetal lung volume was the single best predictor of neonatal survival before fetal endoscopic tracheal occlusion (cutoff 0.23, area under the curve [AUC] 0.88, relative risk 5.3, 95% confidence interval [CI] 1.4-19.7). However, the contralateral lung vascularization index at 4 weeks after fetal endoscopic tracheal occlusion was more accurate in the prediction of neonatal outcome (cutoff 24.0%, AUC 0.98, relative risk 9.9, 95% CI 1.5-66.9) with the combination of observed-to-expected lung volumes and contralateral lung vascularization index at 4 weeks being the best predictor of outcome (AUC 0.98, relative risk 16.6, 95% CI 2.5-112.3). CONCLUSION: Fetal endoscopic tracheal occlusion improves survival rate by increasing the lung size and pulmonary vascularity in fetuses with severe congenital diaphragmatic hernia. The pulmonary response after fetal endoscopic tracheal occlusion can be used to predict neonatal survival. (Obstet Gynecol 2012; 119: 93-101) DOI: 10.1097/AOG.0b013e31823d3aea

Anatomical and functional evaluation of tympanoplasty using a transitory natural latex biomembrane implant from the rubber tree Hevea brasiliensis

PURPOSE: To compare the role of transitory latex and sylastic (R) implants in tympanoplasty on the closure of tympanic perforations. METHODS: A randomized double-blind prospective study was conducted on 107 patients with chronic otitis media submitted to underlay tympanoplasty and divided at random into three groups: control with no transitory implant, latex membrane group, and sylastic (R) membrane group. RESULTS: Greater graft vascularization occurred in the latex membrane group (p<0.05). Good biocompatibility was obtained with the use of the latex and silicone implants, with no effect on the occurrence of infection, otorrhea or otorragy. CONCLUSION: The use of a transitory latex implant induced greater graft vascularization, with a biocompatible interaction with the tissue of the human tympanic membrane.

Dichotomous effects of VEGF-A on adipose tissue dysfunction

Obese fat pads are frequently undervascularized and hypoxic, leading to increased fibrosis, inflammation, and ultimately insulin resistance. We hypothesized that VEGF-A-induced stimulation of angiogenesis enables sustained and sufficient oxygen and nutrient exchange during fat mass expansion, thereby improving adipose tissue function. Using a doxycycline (Dox)-inducible adipocyte-specific VEGF-A overexpression model, we demonstrate that the local up-regulation of VEGF-A in adipocytes improves vascularization and causes a "browning" of white adipose tissue (AT), with massive up-regulation of UCP1 and PGC1 alpha. This is associated with an increase in energy expenditure and resistance to high fat diet-mediated metabolic insults. Similarly, inhibition of VEGF-A-induced activation of VEGFR2 during the early phase of high fat diet-induced weight gain, causes aggravated systemic insulin resistance. However, the same VEGF-A-VEGFR2 blockade in ob/ob mice leads to a reduced body-weight gain, an improvement in insulin sensitivity, a decrease in inflammatory factors, and increased incidence of adipocyte death. The consequences of modulation of angiogenic activity are therefore context dependent. Proangiogenic activity during adipose tissue expansion is beneficial, associated with potent protective effects on metabolism, whereas antiangiogenic action in the context of preexisting adipose tissue dysfunction leads to improvements in metabolism, an effect likely mediated by the ablation of dysfunctional proinflammatory adipocytes.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Three-dimensional Doppler evaluation of single spherical samples from the placenta: intra- and interobserver reliability

Objective To evaluate the intra- and interobserver reliability of assessment of three-dimensional power Doppler (3D-PD) indices from single spherical samples of the placenta. Methods Women with singleton pregnancies at 2440 weeks' gestation were included. Three scans were independently performed by two observers; Observer 1 performed the first and third scan, intercalated by the scan of Observer 2. The observers independently analyzed the 3D-PD datasets that they had previously acquired using four different methods, each using a spherical sample: random sample extending from basal to chorionic plate; random sample with 2 cm3 of volume; directed sample to the region subjectively determined as containing more color Doppler signals extending from basal to chorionic plate; or directed sample with 2 cm3 of volume. The vascularization index (VI), flow index (FI) and vascularization flow index (VFI) were evaluated in each case. The observers were blinded to their own and each other's results. Additional evaluation was performed according to placental location: anterior, posterior and fundal or lateral. Intra- and interobserver reliability was assessed by intraclass correlation coefficients (ICC). Results Ninety-five pregnancies were included in the analysis. All three placental 3D-PD indices showed only weak to moderate reliability (ICC < 0.66 and ICC < 0.48, intra- and interobserver, respectively). The highest values of ICC were observed when using directed spherical samples from basal to chorionic plate. When analyzed by placental location, we found lower ICCs for lateral and fundal placentae compared to anterior and posterior ones. Conclusion Intra- and interobserver reliability of assessment of placental 3D-PD indices from single spherical samples in pregnant women greater than 24 weeks' gestation is poor to moderate, and clinical usefulness of these indices is likely to be limited. Copyright (c) 2012 ISUOG. Published by John Wiley & Sons, Ltd.

Exercise training prevents the microvascular rarefaction in hypertension balancing angiogenic and apoptotic factors role of microRNAs-16,-21, and -126

Aerobic exercise training (ET) lowers hypertension and improves patient outcomes in cardiovascular disease. The mechanisms of these effects are largely unknown. We hypothesized that ET modulates microRNAs (miRNAs) involved in vascularization. miRNA-16 regulates the expression of vascular endothelial growth factor and antiapoptotic protein Bcl-2. miRNA-21 targets Bcl-2. miRNA-126 functions by repressing regulators of the vascular endothelial growth factor pathway. We investigated whether miRNA-16, -21 and -126 are modulated in hypertension and by ET. Twelve-week-old male spontaneously hypertensive rats (SHRs; n=14) and Wistar Kyoto (WKY; n=14) rats were assigned to 4 groups: SHRs, trained SHRs (SHR-T), Wistar Kyoto rats, and trained Wistar Kyoto rats. ET consisted of 10 weeks of swimming. ET reduced blood pressure and heart rate in SHR-Ts. ET repaired the slow-to-fast fiber type transition in soleus muscle and the capillary rarefaction in SHR-Ts. Soleus miRNA-16 and -21 levels increased in SHRs paralleled with a decrease of 48% and 25% in vascular endothelial growth factor and Bcl-2 protein levels, respectively. Hypertension increased Bad and decreased Bcl-x and endothelial NO synthase levels and lowered p-Bad(ser112): Bad ratio. ET in SHR-Ts reduced miRNA-16 and -21 levels and elevated vascular endothelial growth factor and Bcl-2 levels. ET restored soleus endothelial NO synthase levels plus proapoptotic and antiapoptotic mediators in SHR-Ts, indicating that the balance between angiogenic and apoptotic factors may prevent microvascular abnormalities in hypertension. miRNA-126 levels were reduced in SHRs with an increase of 51% in phosphoinositol-3 kinase regulatory subunit 2 expression but normalized in SHR-Ts. Our data show that ET promoted peripheral revascularization in hypertension, which could be associated with regulation of select miRNAs, suggesting a mechanism for its potential therapeutic application in vascular diseases. (Hypertension. 2012;59[part 2]:513-520.). Online Data Supplement

Prediction and probability of neonatal outcome in isolated congenital diaphragmatic hernia using multiple ultrasound parameters

Objectives To evaluate the accuracy and probabilities of different fetal ultrasound parameters to predict neonatal outcome in isolated congenital diaphragmatic hernia (CDH). Methods Between January 2004 and December 2010, we evaluated prospectively 108 fetuses with isolated CDH (82 left-sided and 26 right-sided). The following parameters were evaluated: gestational age at diagnosis, side of the diaphragmatic defect, presence of polyhydramnios, presence of liver herniated into the fetal thorax (liver-up), lung-to-head ratio (LHR) and observed/expected LHR (o/e-LHR), observed/expected contralateral and total fetal lung volume (o/e-ContFLV and o/e-TotFLV) ratios, ultrasonographic fetal lung volume/fetal weight ratio (US-FLW), observed/expected contralateral and main pulmonary artery diameter (o/e-ContPA and o/eMPA) ratios and the contralateral vascularization index (Cont-VI). The outcomes were neonatal death and severe postnatal pulmonary arterial hypertension (PAH). Results Neonatal mortality was 64.8% (70/108). Severe PAH was diagnosed in 68 (63.0%) cases, of which 63 died neonatally (92.6%) (P < 0.001). Gestational age at diagnosis, side of the defect and polyhydramnios were not associated with poor outcome (P > 0.05). LHR, o/eLHR, liver-up, o/e-ContFLV, o/e-TotFLV, US-FLW, o/eContPA, o/e-MPA and Cont-VI were associated with both neonatal death and severe postnatal PAH (P < 0.001). Receiver-operating characteristics curves indicated that measuring total lung volumes (o/e-TotFLV and US-FLW) was more accurate than was considering only the contralateral lung sizes (LHR, o/e-LHR and o/e-ContFLV; P < 0.05), and Cont-VI was the most accurate ultrasound parameter to predict neonatal death and severe PAH (P < 0.001). Conclusions Evaluating total lung volumes is more accurate than is measuring only the contralateral lung size. Evaluating pulmonary vascularization (Cont-VI) is the most accurate predictor of neonatal outcome. Estimating the probability of survival and severe PAH allows classification of cases according to prognosis. Copyright (C) 2011 ISUOG. Published by John Wiley & Sons, Ltd.

Immunohistochemical evaluation of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth

The aim of this study was to evaluate the immunoexpression of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth and to correlate the results with demographic, survival, clinical (TNM staging) and histopathological variables (tumor grade, perineural invasion, embolization and bone invasion). Data from medical records and diagnoses of 41 patients were reviewed. Histological sections were subjected to immunostaining using primary antibodies for human MMP-2, MMP-9 and CD31 and streptavidin-biotin-immunoperoxidase system. Histomorphometric analyses quantified positivity for MMPs (20 fields per slide, 100?points grade, ×200) and for CD31 (microvessels <50?µm in the area of the highest vascularization, 5 fields per slide, 100?points grade, ×400). Statistical design was composed by non-parametric Mann-Whitney U test (investigating the association between numerical variables and immunostainings), chi-square frequency test (in contingency tables), Fisher's exact test (when at least one expected frequency was less than 5 in 2×2 tables), Kaplan-Meier method (estimated probabilities of overall survival) and Iogrank test (comparison of survival curves), all with a significance level of 5%. There was a statistically significant correlation between immunostaining for MMP-2 and lymph node metastasis. Factors associated negatively with survival were N stage, histopathological grade, perineural invasion and immunostaining for MMP-9. There was no significant association between immunoexpression of CD31 and the other variables. The intensity of immunostaining for MMP-2 can be indicative of metastasis in lymph nodes and for MMP-9 of a lower probability of survival