Biological potentials for a family of disintegrin and metalloproteinase (ADAMDEC)-1 in mouse normal pregnancy

Our previous research has indicated local expression of ADAMDEC-1, a family of disintegrin and metalloproteinase, was confirmed in the mouse placentas and enhancement was found in the sites for spontaneous abortion. Present study was aimed to identify biological effects of ADAMDEC-1 in pregnancy process. Syngeneic pairs of C57BL/6J mice and heterogenic mating pairs of CBA/J and DBA/2 mice were used. Pregnant mice were treated with recombinant ADAMDEC-1 protein. Vasculogenesis effects was evaluated using the Matrigel plugs including vascular endothelial growth factor singularity or combination with ADAMDEC-1. ADAMDEC-1 single effects were evaluated by tubal formation and proliferation assays using HuEht-1 endothelial cells. Expression of ADAMDEC-1 was not exactly corresponded with the time periods for miscarriage initiation. ADAMDEC-1 was distributed in normal placentas and fetuses, especially at extraembryonic ectoderm, decidua cells, uterine natural killer (uNK) cells in decidua, trophoblasts in labyrinthine zone, and hematopoietic cells in umbilical blood and fetal liver. ADAMDEC-1 treatment did not affect reproductive performances, while it elevated uNK cell recruitment in placenta and enlarged lumen sizes of the intraplacental vessels. In vitro analysis also indicated ADAMDEC-1 promoting effect on tubal formation and cell length of HuEht-1. qPCR analysis showed that ADAMDEC-1 modified placental gene expression especially for linkage of actin filament rearrangement. Our findings suggested that ADAMDEC-1 is correlated on cell shape, stability, and movement via modification of actin cytoskeleton. ADMADEC-1 suspected to regulate cellular activity of endothelial cells, trophoblasts, and uNK cells and may support normal developing of mouse placentas.     

Electron microscopic cytochemical studies of anionic sites in the rat spleen

The distribution patterns of the intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of endothelial cells (ECs) in blood vessels and reticular cells (RCs) in the splenic cord of the rat spleen were studied by an electron microscopic cytochemical method using polyethyleneimine (PEI) as a cationic probe. Spleens from adult male rats were perfusion-fixed with 0.5% glutaraldehyde -4% paraformaldehyde containing 0.05% cetylpyridinium chloride and then perfused with 0.5% PEI at pH 7.4. On the free surfaces (glycocalyx of the plasma membrane) of the ECs examined, distinct PEI-positive reactions were observed in blood vessels, such as trabecular arteries, central arteries, arterial capillaries, pulp veins and trabecular veins. These PEI-positive electron-dense substances in the trabecular arteries, central arteries, and trabecular veins took the shape of a band of 170-250 nm in thickness. On the other hand, the corresponding ultrastructure of the ECs lining the splenic sinuses and the RCs in the splenic cord showed exceedingly weak PEI reactions. The PEI-reactive deposits were significantly thinner than those in the above blood vessels. As the thickness of the electron-dense substances can be related to the density of the negative charge, these results suggest that there is a high intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of ECs in blood vessels where blood cells and plasma pass into the red pulp or are discharged from the red pulp. In contrast, the splenic sinuses and RCs, which are the main components of the red pulp, contain weakly negative-charged sites. This may contribute to the microcirculation of the splenic blood vessels and elucidate the possible physiological functions of the spleen, such as blood storage.     

Pathogenesis of placentitis in the goat inoculated with Brucella abortus. I. Gross and histologic lesions

Pregnant goats were given Brucella abortus intravenously or in uterine arteries, and tissues from the uterus and placentae were examined at various post-inoculation intervals to study mechanisms of placental infection. Placentitis was present by 5 days post-inoculation and abortions occurred within 11 days. B. abortus was identified in placentae by light microscopy and immunoperoxidase techniques. B. abortus was first seen in erythrophagocytic trophoblasts of the placentome. Subsequently, high numbers of B. abortus were seen in periplacentomal chorioallantoic trophoblasts. Trophoblast necrosis, chorioallantoic ulceration, and large numbers of B. abortus in chorionic villi were present in later stages of infection. These results suggest that entry and replication of B. abortus in trophoblasts precede placentome and fetal infection and that trophoblasts are the source of B. abortus for these tissues. Experimental caprine brucellosis closely resembles bovine and ovine brucellosis and it provides a model to study the intracellular development of B. abortus in trophoblasts.     

Doppler evaluation of maternal and fetal vessels during normal gestation in the bitch

The aim of this work is to evaluate the haemodynamic characteristics of maternal and fetal vessels during normal pregnancy in bitches, using Colour and Pulsed wave Doppler ultrasonography, in order to obtain more information about maternal and fetal circulation. The blood waveforms of the uteroplacental arteries, aorta, caudal cava vein and umbilical cord of the fetuses were recorded weekly in 16 pregnant bitches. Also, the measurements of Peak Systolic, End Diastolic Velocities, Resistance and Pulsatility Indices were carried out. Uteroplacental blood flow was biphasic while the ones of the umbilical artery and aorta were first systolic and then diastolic. The cava showed a typical waveform of venous vessels. During gestation the EDV and PSV of fetal vessels increased (alpha<0.05) while the PI and RI of all vessels examined decreased (alpha<0.05) except for the IP of the Aorta. The Doppler ultrasonography was used to study the characteristics of maternal and fetal vessel flow and their progressive changes during pregnancy. This study can be considered a further contribution in diagnosing and monitoring high-risk pregnancies in Veterinary Medicine.     

Ultrastructural changes related to the lymph node haemorrhages in acute African swine fever

In order to determine the pathogenic mechanisms involved in lymph node haemorrhages in acute African swine fever ( ), eight pigs were inoculated with virus, strain Malawi'83. Lymph node haemorrhages were observed from three days post infection (dpi) onwards, coinciding with ASF virus replication in monocytes and macrophages adjacent to stimulated endothelial cells, phagocytic stimulation of capillary and small-vessel endothelial cells, increase in the number of fenestrations of endothelial cells, and endothelial cell loss, as well as clusters of blood cells and necrotic material beneath the endothelium. Vascular lumina were blocked by platelet plugs and fibrin microthrombi. These phenomena became more marked as the disease progressed. At five dpi, virus replication was also found in circulating neutrophils. At seven dpi, lesions were more intense and were accompanied by virus replication in sinus and capillary endothelial cells, and in other cell populations including pericytes, fibroblasts, smooth muscle fibres and reticular cells. The results obtained in this study suggest that lymph node haemorrhages are related to endothelial stimulation and the onset of disseminated intravascular coagulation. Virus replication in vessel wall cells occurs only in the final stages of the disease and plays a secondary role.     

Pathogenesis of placentitis in the goat inoculated with Brucella abortus. II. Ultrastructural studies

Pregnant goats were inoculated intravenously or in uterine arteries with Brucella abortus, and tissues from the uterus and placenta were examined by electron microscopy. Identification of B. abortus in placentae was with antibody-coated colloidal gold. B. abortus was first seen in phagosomes of erythrophagocytic trophoblasts and in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Subsequently, trophoblast necrosis and ulceration of chorioallantoic membranes were present. Coincidently, B. abortus was present in the lumen of placental capillaries. In late stages of infection, placental vasculitis was present, and placentomal trophoblasts were separated from maternal syncytial epithelium. In lesions with vasculitis, large numbers of B. abortus were in connective tissue of chorionic villi. Within the placentome, trophoblasts that lined chorionic villi contained no intracellular bacteria and were separated from B. abortus by intact basement membranes. These results suggest that bacteremic B. abortus is endocytosed by erythrophagocytic trophoblasts and that B. abortus replicates in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Replication of brucellae in trophoblastic rough endoplasmic reticulum is unique; we believe that B. abortus may utilize endoplasmic reticulum for synthesis and glycosylation of bacterial membrane proteins or that B. abortus catabolizes trophoblast secretory proteins.     

Vascular lesions in lungs of Bali cattle with Jembrana disease.

Jembrana disease is an acute infectious disease of unknown etiology enzootic among Bali cattle (Bos javanicus) in Indonesia. Morphologic examination of 75 female Bali cattle between 18 months and 4 years old affected with Jembrana disease consistently revealed pulmonary granulomatous vascular lesions. The lesions were diffusely distributed throughout the lung. The principal lesion was the presence of a large number of intravascular macrophages that filled the lumina of pulmonary veins and pulmonary arteries of a vascular diameter of 20-200 microns, excluding the rest of blood cellular components. Concentric layers of perithelial cells also with plasma cells and macrophages were occasionally present around both veins and arteries. Infiltration of polymorphonuclear leukocytes or small lymphocytes was not seen. Destruction or necrosis of tissues or blood vessels was rarely seen. Because this vascular lesion was found in the lungs of all affected cattle examined, this change is useful for the postmortem diagnosis of Jembrana disease. Moreover, its presence could be used to distinguish Jembrana disease from malignant catarrhal fever and other lymphoreticular proliferative conditions that are frequently found among cattle in Indonesia.     

山羊胚胎大动脉壁发育的组织学研究

本文应用光镜和透射电镜对山羊胚胎大动脉壁的发生发育过程进行了系统观察。结果表明：血管发生初期仅由一层内皮细胞构成。随着胚胎的发育，除毛细血管外，其它血管壁形成分层结构，形成内膜、中膜、外膜；胚胎大动脉内皮细胞胞质内存在着丰富的粗面内质网和游离核糖体，代谢是很活跃的；大动脉管壁的内弹性膜形成较晚，先产生小片段，然后呈连续带状；大动脉内膜和中膜内不含典型的成纤维细胞，在中膜内存在一种能产生胶原纤维的细胞，其结构类似于平滑肌细胞。     

Vascular remodeling and its role in the pathogenesis of ascites in fast growing commercial broilers

This study examined the putative role of blood vessel pathology in the development of ascites in broilers. Major blood vessels (aorta, brachiocephalic arteries, pulmonary arteries, and vena cava) from normal commercial male broiler chickens, and broilers that developed congestive heart failure (CHF) with or without ascites were subjected to gross and microscopic examination.On cross-section, grossly, the arteries from normal broilers and those showing dilated cardiomyopathy without ascites appeared circular, with firm wall tone characteristic of the normal artery. In contrast, the arteries from ascitic broilers appeared flaccid and lacked elasticity, which was evidenced by collapsing, ellipsoid cross-sectional arterial lumen owing to the structural weakness of the arterial walls. Microscopically, ascitic broilers showed thinning or occasionally total loss of elastic elements in the arterial wall, and reduced network density of the structural matrix of the vascular wall, as well as increased thickness of fibers in vena cava.The structural changes seen in the major arteries from ascitic broilers are maladaptive, and as such would definitively impose an increased hemodynamic burden on the already failing heart pump. The changes in veins are indicative of pathological remodeling conducive to increased permeability of the vascular wall, particularly in the situation when a poorly distensible structure is further subjected to wall stress associated with increased pressure and volume overload. Taken together, increased hemodynamic burden and reduced structural density of the venous wall constitute conditions conducive for seepage and accumulation of ascitic fluid.     

Early arterial injury-induced myointimal proliferation in canine pulmonary arteries

Transmission electron microscopy was used to study the early vascular response induced by arterial damage with heartworm infection. The pulmonary arteries were examined in dogs 4 days after experimental transplantation of 6 to 8 Dirofilaria immitis adults. Evan's blue dye was given IV and followed in 60 minutes by perfusion fixation with 1% glutaraldehyde. Endothelial cell junctions were disrupted and rounded endothelial cells were observed. Macrophages and neutrophils adhered to abnormal endothelial cells. Focal areas had platelet aggregates adhered to exposed subendothelial structures. Platelets were activated and degranulated. Areas of the internal elastic lamina appeared to be disrupted, and smooth muscle cell processes from the tunica media extended through these regions of disruption. Smooth muscle cells were oriented toward the arterial lumen and some had migrated to the surface. These findings are compatible with the response to injury theory of myointimal proliferation.     

Changes in expression and localization of GPRC5B and RARalpha in the placenta and yolk sac during middle to late gestation in mice

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac.     

Development of the navicular bone in foetal and young horses, including the arterial supply

A macroscopic, arteriographic and histological study of the development and the arterial anatomy of the navicular bone of 33 foetuses and 55 young horses is described. After 125 days of gestation the blood supply consists of two routes: one situated in the superficial layer of the fibrocartilage and the other similar to the blood supply of the navicular bone of the normal mature horse. After 270 days gestation, the blood vessels in the fibrocartilage gradually regressed and retracted until they have disappeared at six months after birth. At two months after birth the first macroscopic thinning of the fibrocartilage was noticed. From seven months to one year about 45 per cent of the navicular bones showed a slight thinning of the fibrocartilage. A positive correlation was found between radiographic abnormalities (ie enlargement of the nutrient foramina) and the frequency of thinning of the fibrocartilage. Radiographic abnormalities were first recognised 14 days after birth, whereas the arteriogram showed the first changes such as fewer or no arteries entering distally at the distal extremities at 10 weeks after birth. At four weeks after birth the first arterial wall changes were found, ie intimal thickening with or without splitting of the internal elastic membrane. From that age onward, the number of navicular bones with arterial wall changes gradually increased. Starting at five months after birth only 6 to 20 per cent of the arteries in the navicular bones without radiographic abnormalities showed arterial wall changes. However, the navicular bones with radiographic abnormalities showed arterial wall changes in 25 to 80 per cent of the arteries.     

Development of transferred xenogeneic vole embryos in mouse uteri

An experimental model to study interspecific pregnancy using voles, Microtus arvalis, and green fluorescent protein gene‐induced transgenic mice is presented. Xenogeneic blastocysts from the vole were transferred into the uteri of pseudopregnant mice along with allogeneic blastocysts from green fluorescent protein gene‐induced transgenic mice. The uteri containing xeno‐allo combined transfers were examined from day 6 to 13 of gestation. Although the vole embryos implanted, the uteri containing vole embryos were smaller compared with those having allogeneic mouse embryos. On day 8, the uteri containing vole embryos hemorrhaged internally and no vole embryo was found in the pregnant uterus after day 11. Allogeneic mouse embryos developed normally despite the presence and abortion of the vole embryos. In uteri implanted with vole embryos, decidua were formed and numerous blood vessels were distributed around the embryo. Maternal blood cells infiltrated into the celomic cavity of the vole embryo through the discontinuous region of trophoblast. Periodic acid‐Schiff‐positive granulated metrial gland cells were remarkably increased in the decidual sites. These findings suggest that a disorder of embryo–maternal interaction might induce the appearance of numerous granulated metrial gland cells and rejection of the embryos.     

Quantitative expression and immunohistochemical detection of glucose transporters, GLUT1 and GLUT3 in the rabbit placenta during successful pregnancy

Glucose is essential for the development of the fetus. We address here the quantitative expression and immunohistochemical localization of glucose transporter (GLUT1 and GLUT3) in the rabbit placenta during successful pregnancy. Blood glucose level showed a significant decrease at the gestation period in comparison with non-pregnancy. Maternal serum glucose was gradually increased according to fetal development. Quantitative RT-PCR results showed that expression of GLUT1 was significantly increased from day 13 to day 18, while GLUT3 mRNA level was significantly decreased during the same periods. Western blot analysis demonstrated that GLUT1 protein did not change significantly in the placenta during pregnancy when compared to non-pregnant uteri. Immunohistochemistry indicated that distribution of GLUT1 was observed mainly to the surface of the outer trophoblasts, whereas GLUT3 mainly localized to the basal site of the inner trophoblasts and fetal blood vessels. These results suggest that glucose is transported through GLUT1 from the maternal blood stream for use as a placental fuel and for further transport through GLUT3 to the fetal circulation, thus signifying the distinct anatomical localization of GLUT1 and GLUT3 in the rabbit placenta during successful pregnancy.     

Labelling of rat endothelial cells with antibodies to vWF, RECA-1, PECAM-1, ICAM-1, OX-43 and ZO-1

Labelling with endothelium specific monoclonal antibodies, von Willebrand Factor (vWF), rat endothelial cell antigen-1 (RECA-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), OX-43 and zonula occludentes-1 (ZO-1), was investigated in cryostat sections of vessels from rats of different ages using a confocal microscope. The results showed that labelling of the vWF was positive in endothelial cells from adult, fetal and different ages of embryonic rat. Labelling with RECA-1 was weakly positive in adult rat aorta and lung endothelial cells but not in embryonic yolk sac endothelial cells. Labelling using PECAM-1, ICAM-1 and OX-43 was negative in both adult and embryonic endothelial cells. ZO-1 showed positive but very weak reactivity in embryonic yolk sac endothelial cells. The expression of vWF on vessels from adult and 19.5-day fetal tissues was strongly positive. However, the expression of vWF in embryonic endothelial cells was dependent on the gestational age. While the 11.5-day yolk sac vessels stained weakly, staining gradually increased in 13.5-, 15.5- and 17.5-day-old yolk sac vessels. The results suggest that vWF is a reliable endothelial cell marker in rat vascular endothelial cells, including both fetal and embryonic stages.     

Histomorphologic structure of the carotid rete-cavernous sinus complex and its functional importance in sheep (Ovis aries)

Paraffin sections of the carotid rete-cavernous sinus complex of sheep were studied, using different stains. The carotid rete of sheep was composed of medium-sized arteries with smooth muscle layers that were oriented in different directions. The carotid body cells may have migrated proximally in the adventitia of the intracranial portion of the internal carotid artery as its extracranial portion degenerates early in life. The cavernous sinus shared a common tunica adventitia with surrounding rete branches. At places, the wall of the cavernous sinus had a distinct tunica media interposed between the endothelial cells and the tunica adventitia. Therefore, the name cavernous venous plexus has been proposed for the cavernous sinus in sheep.     

The pathogenesis of leptospirosis I. Hemorrhages in experimental leptospirosis in guinea pigs.

In experimental infections of guinea pigs with a virulent strain of Leptospira icterohaemorrhagiae widespread hemorrhages were observed. Thrombocytopenia, prolongation of prothrombin, thrombin, partial thromboplastin and coagulation times, decrease of plasma fibrinogen, factor V, factor VIII and the presence of fibrinogen degradation products were demonstrated. Treatment of infected guinea pigs with heparin prolonged life for two to three days. The histological observations revealed that the main lesion is a severe injury of the vasculature, mainly arteries, arterioles and capillaries. Most of the endothelial cells are affected or destroyed and the muscular fibers of arteries and arterioles are injured. With Martius-Scarlet-Blue, Weigert or Picro-Mallory stains it was demonstrated that the organization seen in the vessels is not all made of fibrin. The conclusion reached was that the hemorrhages observed in experimental leptospirosis in guines pigs are due to disseminated intravascular coagulation.