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.

Histomorphometrical and proliferative aspects of placenta and uterus of the collared peccary (Tayassu tajacu)

The histomorphometric and proliferative characteristics of the collared peccary (Tayassu tajacu) placenta and uterus were analyzed. The material was examined by standard histological techniques and histochemistry (PAS, Perls and Alcian Blue pH 0.5 and 2.5%) and the cellular proliferation by AgNORs and flow cytometry. All the analyzed morphometric variables differed between pregnant and non-pregnant uteri in the luteal phase using the Dunnet test. Height and gland diameter of uterine glands increased linearly during pregnancy, with an intense positive PAS and Perls reaction in all stages. The cells with more than seven AgNORs per nuclei and the cells in the G2M cell cycle phase in the maternal tissue also increased after 70 days of pregnancy. The uteroplacental ridges had a linear increase in size with two distinct areas, base and top, with uterine epithelium and trophoblastic cells changing their morphology following the placental ridge development. Flow cytometry analysis showed the percentage of cells in each cell cycle phase with a quadratic behavior for stages G2/M in the maternal tissue, suggesting an increase in proliferative capacity of maternal tissue after 65 days of pregnancy. The same quadratic effect was observed in the G0/G1 phase in both maternal and fetal tissues. Cells in apoptosis showed cubic behavior in both tissues. The morphometric and cellular dynamic aspects observed in this study have not been previously described and they extend our knowledge of functions relating to maternal-fetal dynamics in this species.

IMMUNE-ANGIOGENESIS MECHANISMS ASSOCIATED WITH PORCINE PREGNANCY SUCCESS AND FAILURE

Spontaneous fetal loss (25-40%) leading to decrease in litter size is a significant concern to the pork industry. A deficit in the placental vasculature has emerged as one of the important factors associated with fetal loss. During early pig pregnancy, the endometrium becomes enriched with immune cells recruited by conceptus-derived signals including specific chemokine stimuli. These immune cells assist in various aspects of placental development and angiogenesis. Recent evidence suggests that microRNAs (miRNAs: small non-coding RNAs that regulate gene expression) regulate immune cell development and their functions. In addition, intercellular communication including exchange of biomolecules (e.g. miRNAs) between the conceptus and endometrium regulate key developmental processes during pregnancy. To understand the biological significance of immune cell enrichment, regulation of their functions by miRNAs and transfer of miRNAs across the maternal fetal-interface, we screened specific sets of chemokines and pro- and anti-angiogenic miRNAs in endometrial lymphocytes (ENDO LY), endometrium, and chorioallantoic membrane (CAM) isolated from conceptus attachment sites (CAS) during early, gestation day (gd)20 and mid-pregnancy (gd50). We report increased expression of selected chemokines including CXCR3 and CCR5 in ENDO LY and CXCL10, CXCR3, CCL5, CCR5 in endometrium associated with arresting CAS at gd20. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5, and CCR5) in endometrium and CAM. We report for the first time significant differences for miRNAs involved in immune cell-derived angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between ENDO LY associated with healthy and arresting CAS. Significant differences were also found in endometrium and CAM for some miRNAs (miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, we confirm that placenta specific-exosomes contain proteins and 14 select miRNAs including miR-126-5P, miR-296-5P, miR-16, and miR-17-5P that are of relevance to early implantation events. We further demonstrated the bidirectional exosome shuttling between porcine trophectoderm cells (PTr2) and porcine aortic endothelial cells (PAOEC). PTr2-derived exosomes were able to modulate the endothelial cell proliferation that is crucial for the establishment of pregnancy. Our data unravels the selected chemokines and miRNAs associated with immune cell-regulated angiogenesis and reconfirm that exosome mediated cell-cell communication opens-up new avenues to understand porcine pregnancy.

Porcine skin as a source of biodegradable matrices: alkaline treatment and glutaraldehyde crosslinking

In this work, the modifications promoted by alkaline hydrolysis and glutaraldehyde (GA) crosslinking on type I collagen found in porcine skin have been studied. Collagen matrices were obtained from the alkaline hydrolysis of porcine skin, with subsequent GA crosslinking in different concentrations and reaction times. The elastin content determination showed that independent of the treatment, elastin was present in the matrices. Results obtained from in vitro trypsin degradation indicated that with the increase of GA concentration and reaction time, the degradation rate decreased. From thermogravimetry and differential scanning calorimetry analysis it can be observed that the collagen in the matrices becomes more resistant to thermal degradation as a consequence of the increasing crosslink degree. Scanning electron microscopy analysis indicated that after the GA crosslinking, collagen fibers become more organized and well-defined. Therefore, the preparations of porcine skin matrices with different degradation rates, which can be used in soft tissue reconstruction, are viable.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Avaliação morfológica das membranas fetais e da placenta de Mazama gouazoubira (veado-catingueiro) de vida livre no terço inicial da gestação

Mazama gouazoubira, o veado-catingueiro, é uma espécie de cervídeo de porte pequeno, que pode ser encontrado na América do Sul, desde o sul do Uruguai até o norte de Mato Grosso, no Brasil. Este estudo teve como objetivo descrever as membranas fetais e a placenta de M. gouazoubira no terço inicial de gestação. As amostras coletadas foram analisadas macroscopicamente e microscopicamente. O exame do útero demonstrou uma gestação univitelina e um embrião com crown-rump de 13mm. Na análise do embrião pode ser observado o olho pigmentado, as saliências auriculares, o mesonefro e e metanefro em desenvolvimento, o fígado e sua proeminência externa, o estômago, os membros torácicos e os brotos dos membros pélvicos. A placenta apresentou-se oligocotiledonária e no útero puderam ser observadas nove carúnculas. O saco gestacional mediu 15cm de comprimento e, como observado no início da gestação dos ruminantes domésticos, os cotilédones não puderam ser identificados macroscopicamente. Uma fraca adesão foi observada entre as carúnculas e a membrana corioalantóica (cotilédones) que formavam os placentônios. A membrana corioalantóica demonstrou um alantóide bem vascularizado composto por uma fina camada de células de núcleos e citoplasmas alongados. Na outra face da membrana, o cório foi composto por células cúbicas, de citoplasmas escassos e núcleos grandes e arredondados, características de células trofoblásticas. Envolvendo o embrião visualizou-se o saco amniótico constituído de duas camadas passíveis de separação mecânica, que apresentaram morfologia similar, sendo formadas por um epitélio pavimentoso avascular. Não foi observado saco vitelino no estágio gestacional do espécime estudado. Conclui-se que a placenta de M. gouazoubira é oligocotiledonária, como observado em outros cervídeos, e que as membranas fetais apresentam semelhanças com a de outros ruminantes, incluindo as características citológicas. Estudos adicionais são necessários para determinar a presença do saco vitelino e quando ocorre sua regressão.

Molecular basis for porcine parvovirus detection in dead fetuses

Reproductive failures are still common grounds for complaint by commercial swine producers. Porcine parvovirus (PPV) is associated with different clinical reproductive signs. The aim of the present study was to investigate PPV fetal infection at swine farms having ongoing reproductive performance problems. The presence of virus in fetal tissues was determined by nested-polymerase chain reaction assay directed to the conserved NS1 gene of PPV in aborted fetuses, mummies and stillborns. Fetuses show a high frequency of PPV infection (96.4%; N = 28). In 60.7% of the fetuses, PPV were detected in all tissue samples (lung, heart, thymus, kidney, and spleen). Viral infection differed among fetal tissues, with a higher frequency in the lung and heart (P < 0.05). Fetuses with up to 99 days of gestational age and from younger sows showed a higher frequency of PPV (P < 0.05). No significant difference in the presence of PPV was detected among the three clinical presentations. The results suggest that PPV remains an important pathogenic agent associated with porcine fetal death.

Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.

Maternal diabetes affects cell proliferation in developing rat placenta

Placentation starts with the formation of a spheroidal trophoblastic shell surrounding the embryo, thus facilitating both implantation into the uterine stroma and contact with maternal blood. Although it is known that diabetes increases the placental size and weight, the mechanisms responsible for this alteration are still poorly understood. In mammals, cellular proliferation occurs in parallel to placental development and it is possible that diabetes induces abnormal uncontrolled cell proliferation in the placenta similar to that seen in other organs (e.g. retina). To test this hypothesis, the objective of this work was to determine cell proliferation in different regions of the placenta during its development in a diabetic rat model. Accordingly, diabetes was induced on day 2 of pregnancy in Wistar rats by a single injection of alloxan (40 mg/kg i.v.). Placentas were collected on days 14, 17, and 20 postcoitum. Immunoperoxidase was used to identify Ki67 nuclear antigen in placental sections. The number of proliferating cells was determined in the total placental area as well as in the labyrinth, spongiotrophoblast and giant trophoblast cell regions. During the course of pregnancy, the number of Ki67 positive cells decreased in both control and diabetic rat placentas. However, starting from day 17 of pregnancy, the number of Ki67 positive cells in the labyrinth and spongiotrophoblast regions was higher in diabetic rat placentas as compared to control. The present results demonstrate that placentas from the diabetic rat model have a significantly higher number of proliferating cells in specific regions of the placenta and at defined developmental stages. It is possible that this increased cell proliferation promotes thickness of the placental barrier consequently affecting the normal maternal-fetal exchanges.

Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

Background: Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy. Methods: Mif was immunolocalized at implantation sites on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. Ectoplacental cones and fetal placentas dissected from the maternal tissues were used for Western blotting and qRT-PCR assays on the same gestation days. Results: During the post-implantation period (gd7.5), trophoblast giant cells showed strong Mif reactivity. In later placentation phases (gds 10.5-17.5), Mif appeared to be concentrated in the junctional zone and trophoblast giant cells. Mif protein expression increased significantly from gd7.5 to 10.5 (p = 0.005) and from gd7.5 to 13.5 (p = 0.03), remaining at high concentration as gestation proceeded. Higher mRNA expression was found on gd10.5 and was significantly different from gd13.5 (p = 0.048) and 17.5 (p = 0.009). Conclusions: The up-regulation of Mif on gd10.5 coincides with the stage in which the placenta assumes its three-layered organization (giant cells, spongiotrophoblast and labyrinth zones), fetal blood circulation begins and population of uNK cells reaches high proportions at the maternal counter part of the placenta, suggesting that Mif may play a role in either the placentation or in the adaptation of the differentiated placenta to the uterus or still in gestational immunomodulatory responses. Moreover, it reinforces the possibility of specific activities for Mif at the maternal fetal interface.

Precipitation of Porcine Insulin With Carbon Dioxide

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO(2)) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO(2) as an acid- precipitating agent. The Solubility of porcine insulin in NaHCO(3) solutions in pressurized CO(2) was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoclectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self-association reactions were also taken into account. The biological activity of insulin was maintained after precipitation With CO(2), although some activity can be lost if foam is formed in the depressurization step. Biotechnol. Bioeng. 2009;103: 909-919. (C) 2009 Wiley Periodicals, Inc.

Excised Porcine Cornea Integrity Evaluation in an in vitro Model of Iontophoretic Ocular Research

Background/Aims: It is a challenge to adapt traditional in vitro diffusion experiments to ocular tissue. Thus, the aim of this work was to present experimental evidence on the integrity of the porcine cornea, barrier function and maintenance of electrical properties for 6 h of experiment when the tissue is mounted on an inexpensive and easy-to-use in vitro model for ocular iontophoresis. Methods: A modified Franz diffusion cell containing two ports for the insertion of the electrodes and a receiving compartment that does not need gassing with carbogen was used in the studies. Corneal electron transmission microscopy images were obtained, and diffusion experiments with fluorescent markers were performed to examine the integrity of the barrier function. The preservation of the negatively charged corneal epithelium was verified by the determination of the electro-osmotic flow of a hydrophilic and non-ionized molecule. Results: The diffusion cell was able to maintain the temperature, homogenization, porcine epithelial corneal structure integrity, barrier function and electrical characteristics throughout the 6 h of permeation experiment, without requiring CO(2) gassing when the receiving chamber was filled with 25 m M of HEPES buffer solution. Conclusion: The system described here is inexpensive, easy to handle and reliable as an in vitro model for iontophoretic ocular delivery studies. Copyright (C) 2010 S. Karger AG, Basel

Perioperative Temporary Occlusion of the Internal Iliac Arteries as Prophylaxis in Cesarean Section at Risk of Hemorrhage in Placenta Accreta

Purpose The purpose of this study was to describe the preliminary results of prophylactic temporary balloon occlusion of the internal iliac arteries for bleeding control in patients with placenta accreta during cesarean hysterectomy. Methods From May 2006 to March 2010, 21 patients diagnosed with placenta accreta using ultrasound and/or magnetic resonance imaging were submitted to prophylactic balloon occlusion before hysterectomy. Fluoroscopy, balloon occlusion time, surgical duration, intraoperative blood loss, transfusion volume, and procedure complications were analyzed. Results The mean age was 30.5 years with a mean of 3.6 previous gestations. Imaging studies revealed that all patients had placenta accreta and all were submitted to cesarean hysterectomy. One hysterectomy was due to previous diagnosis of fetal death and another due to cesarean with uterine curettage. Mean fluoroscopy time was 7.5 min, balloon occlusion time was 164 min, and surgery duration was 260 min. Estimated blood loss was 1,671.5 ml with mean reposition fluids of 3,538 ml of crystalloids, 309.5 ml of colloids, and 1.24 ml of packed red blood cells. Two patients were submitted to thromboembolectomy due to prolonged surgical time. There was no maternal or fetal mortality related to the procedure. Conclusions The results demonstrated that prophylactic balloon occlusion of internal iliac artery is a safe method and appears to reduce blood loss and transfusion requirements in patients diagnosed with placenta accreta who undergo cesarean hysterectomy. Antenatal imaging diagnosis of placenta accreta enables preoperative planning.

Impact of Stent-Graft Oversizing on the Thoracic Aorta: Experimental Study in a Porcine Model

Purpose: To analyze in an experimental animal model the effect of 4 different levels of stents-graft oversizing on non-atherosclerotic aortas such as those found in young individuals who undergo stent-graft repair for traumatic aortic injuries. Methods: The diameter of the porcine thoracic aorta is similar to the aorta of young adults (18-20 mm), so 25 pigs were randomized into 5 groups: 1 control (without stent-graft) and 4 oversizing groups (A: 10%-19%, B: 20%-29%, C: 30%-39%, and D: >40%). Two types of biomechanical tests were performed on all aortas 4 weeks after endoprosthesis deployment. Results: The results of the detachment test, which analyzed the strength necessary to remove the stent-graft from the aorta, were similar in the 4 groups (A: 42 N, B: 41 N, C: 46 N, and D: 46 N). However, 2 aortas ruptured during the tests (groups C and D). The second test was performed in 3 aortic segments. Maximum shear strength, maximum stress, and maximum tension supported by the aortic wall had a negative and linear correlation with oversizing. There were significant differences in all 4 groups when compared with the control group. Strain, which reflects the elastic properties of the aortic wall, was very similar in all 4 groups, but a great difference was found when compared with the control group (p<0.0001). Conclusion: The study showed an important subacute change in the biomechanical properties of the aortic wall after implantation of an oversized endoprosthesis. This weakness of the aortic wall was confirmed by 2 ruptures during the detachment test. These results partially explain the interaction of stent-grafts with non-atherosclerotic thoracic aortas and may serve as a basis for further studies and the development of specific material to be used in vascular trauma and young patients. J Endovasc Ther. 2011; 18: 576-584

Particulate urban air pollution affects the functional morphology of mouse placenta

in humans, adverse pregnancy outcomes (low birth weight, prematurity, and intrauterine growth retardation) are associated with exposure to urban air pollution. Experimental data have also shown that such exposure elicits adverse reproductive outcomes. We hypothesized that the effects of urban air pollution on pregnancy outcomes could be related to changes in functional morphology of the placenta. To test this, future dams were exposed during pregestational and gestational periods to filtered or nonfiltered air in exposure chambers. Placentas were collected from near-term pregnancies and prepared for microscopical examination. Fields of view on vertical uniform random tissue slices were analyzed using stereological methods. Volumes of placental compartments were estimated, and the labyrinth was analyzed further in terms of its maternal vascular spaces, fetal capillaries, trophoblast, and exchange surface areas. From these primary data, secondary quantities were derived: vessel calibers (expressed as diameters), trophoblast thickness (arithmetic mean), and total and mass-specific morphometric diffusive conductances for oxygen of the intervascular barrier. Two-way analysis of variance showed that both periods of exposure led to significantly smaller fetal weights. Pregestational exposure to nonfiltered air led to significant increases in fetal capillary surface area and in total and mass-specific conductances. However, the calibers of maternal blood spaces were reduced. Gestational exposure to nonfiltered air was associated with reduced volumes, calibers, and surface areas of maternal blood spaces and with greater fetal capillary surfaces and diffusive conductances. The findings indicate that urban air pollution affects placental functional morphology. Fetal weights are compromised despite attempts to improve diffusive transport across the placenta.