Short and long-term repercussions of the experimental diabetes in embryofetal development.

BACKGROUND: Diabetic pregnancy have increased rates of congenital malformation and neonatal mortality. In vitro studies suggest hyperglycemia associated with diabetes impair embryogenesis but in vivo investigations on maternal hyperglycemic insult and early embryo development are scarce. We evaluated the embryofetal development on experimental diabetes models to assess whether hyperglycemia at preimplantation period impairs the progression of pregnancy. METHODS: Different hyperglycemic intensities were obtained by two experimental diabetes models. Female Sprague Dawley rats received streptozotocin at birth (mild diabetes) or at day 90 of life (severe diabetes). For both diabetic groups hyperglycemia was confirmed 5 days after diabetes induction and the mating was performed around 100 day of life. For preimplantation analysis, embryos were recovered at D4 of pregnancy. Another group of animals was submitted to laparotomy at D21 to assess contents of the uterus and fetal viability. RESULTS: Mild (i) and Severe (ii) diabetes modified the early development. Degenerating embryos percentage was higher compared to control (11%) (i) 30.7%, (ii) 37.3%. Cell number mean dropped according to hyperglycemic intensity (control 30.57, (i) 21.42, (ii) 13.42). Pre and post-implantation loss rates were higher in diabetic groups. The fetal viability also decreased from 96% in the control group to (i) 78.7% and (ii) 80.6%. CONCLUSION: Our results show that during diabetic pregnancy, preimplantation embryos present decreased cell number due to higher apoptosis rates, which are dependent of the hyperglycemic intensity. Moreover, fetal viability was also decreased suggesting that the quality of these embryos at long-term may be questioned.

Influence of culture conditions on the secretome of mesenchymal stem cells derived from feline adipose tissue: Proteomics approach.

This study aimed to describe the secretome of mesenchymal stem cells derived from feline adipose tissue (AD-MSCs) and compare the effects of different culture conditions on AD-MSC proteomics using a shotgun approach. Adipose tissue was collected from 5 female cats and prepared to culture. Conditioned media was collected at third passage, in which the cells were cultured under 4 conditions, normoxia with fetal bovine serum (N + FBS), hypoxia with FBS (H + FBS), normoxia without FBS (N - FBS), and hypoxia without FBS (H - FBS). Then, the secretome was concentrated and prepared for proteomic approaches. Secretomes cultured with FBS-free medium had more than twice identified proteins in comparison with the secretomes cultured with FBS. In contrast, hypoxic conditions did not increase protein amount and affected only a small proteome fraction. Relevant proteins were related to the extracellular matrix promoting environmental modulation, influencing cell signaling pathways, and providing a suitable environment for cell proliferation and maintenance. Moreover, other proteins were also related to cell adhesion, migration and morphogenesis. Culture conditions can influence protein abundance in AD-MSC secretome, and can give also more specificity to cell and cell-free treatments for different diseases.

Intrathecal Transplantation of Autologous and Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells in Dogs.

The route used in the transplantation of mesenchymal stem cells (MSCs) can directly affect the treatment success. The transplantation of MSCs via the intrathecal (IT) route can be an important therapeutic strategy for neurological disorders. The objective of this study was to evaluate the safety and feasibility of the IT transplantation of autologous (Auto-MSCs) and allogeneic (Allo-MSCs) bone marrow mesenchymal stem cells (BM-MSCs) in healthy dogs. Based on neurodisability score, cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI), no significant differences from the control group were observed on day 1 or day 5 after IT Auto- or Allo-MSCs transplantation (P > 0.05). In addition, analysis of matrix metalloproteinase (MMP)-2 and MMP-9 expression in the CSF revealed no significant differences (P > 0.05) at 5 days after IT transplantation in the Auto- or Allo-MSCs group when compared to the control. Intrathecal transplantation of BM-MSCs in dogs provides a safe, easy and minimally invasive route for the use of cell-based therapeutics in central nervous system diseases.

PP100. Pre-eclampsia: Risk factors and outcomes - A two-year study.

INTRODUCTION: Pre-eclampsia (PE) is a hypertensive disorder responsible for major morbidity and mortality in both mother and fetus. There are some risk factors associated with this entity, but it remains very difficult to predict. OBJECTIVES: Study the incidence of PE and the related risk factors, as well as the maternal and fetal outcome. METHODS: We reviewed the clinical records of pregnant women admitted to Prof. Fernando Fonseca's Hospital from January 2008 to December 2009, with the diagnosis of pre-eclampsia. The statistic analysis was based on Excel 2007. RESULTS: There were 90 cases of PE, among the 308 hypertensive disorders reviewed, with an incidence of 1,1% in overall population of pregnant women. Risk factors with higher association were Chronic Hypertension before pregnancy (24,4%), maternal age above 35 years old (16,67%), maternal age under 20 years old (14,44%), and previous episode of pre-eclampsia (8,89%). Major maternal complications that determined Intensive Care Unit admission were recorded in 17 cases (18,89%), with 3 HELLP syndromes (Hemolysis, elevated liver enzymes, and low platelets)(3,33%). No maternal death was recorded. Preterm delivery (PTD) was seen in 61,1%, 32% before 34weeks and 6,67% before 28weeks. There were 19 cases of 1st minute Apgar Index below 7 and 5 cases of 5th minute Apgar Index below 7. There was one in utero death and two interruptions of pregnancy below 24 weeks due to serious PE. Three twin pregnancies. CONCLUSIONS: PE is a form of hypertensive pregnancy disorder, with a risk of recurrence in subsequent pregnancies. It has a catastrophic potential, mainly associated to PTD, and also with significant morbidity to the pregnant women, reflected in the incidence of admissions to ICU, HELLP syndrome and end-organ failure. In our study we confirmed the adverse outcomes related to this entity, and the risk factors associated.

PP099. Chronic hypertension: follow-up of chronic hypertension pregnant women in 2008-2009.

INTRODUCTION: Chronic Hypertension (CH) is one of the most prevalent diseases in the world. Because women are getting pregnant in late ages, the incidence of Chronic Hypertension in pregnancy is raising (2-3%). As a long term disease in its essential form, with low repercussion in target-organs at reproductive age, the medical complications related to CH are not the main concern of the pregnant women. However, it influences the pregnancy and may result in serious maternal-fetal complications, especially if a preeclampsia/eclampsia superimposes to pregnancy. The complications may be manifested as placental abruption, acute renal failure, cardiac decompensation, and cerebral accidents in the mother and of growth restriction and unexplained mid-trimester fetal death. The main goal in the follow-up of this pregnant women is to achieve controlled levels of blood pressure (Systolic - 110-140mmHg, Diastolic - 80-90mmHg) with anti-hypertensive drugs, and evaluate and early diagnose the most serious complication, preeclampsia/eclampsia(PE/E). OBJECTIVES: Review the cases of Chronic Hypertension followed in our institution in 2008-2009, with the analysis of population, anti-hypertensive drugs required to control HTA, fetal complications (growth restriction, preterm delivery and fetal death) and maternal/fetal complications (PE/E, Abruption placentae, Renal and Cardiac acute insufficiency, and cerebral accidents). METHODS: In a retrospective study, from January 2008 to December 2009, were analysed all files related with Chronic Hypertension followed in the obstetric department of Prof. Fernando Fonseca's Hospital. The statistic analysis was based on Excel 2007. RESULTS: The incidence of Chronic Hypertension was 40% of all hypertensive disorders in this period and of 1,5% in overall pregnant population. The maternal mean age of this group was 33,8 years (16;44) being the most prevalent pre-existing co-morbidities the endocrinologic disorders (obesity, Diabetes and thyroid pathology). 38% were previous medicated with anti-hypertensive drugs, the majority of them needing only one drug to control blood pressure (BP) (64%). During the pregnancy follow up, 78% were medicated with one or more anti-hypertensive drugs, requiring only one drug to control BP in the majority of cases 66%. In terms of fetal complications 3 cases of growth restriction were signed (2%), preterm delivery in 26 cases (21%) and 2 interruptions of pregnancy in the second trimester before 24 weeks because of maternal complications (2%). There was one case of Abruption Placentae (1%). In terms of maternal complications there were 5 ICU internments (4%), and one situation of acute cardiac disfunction (1%). The CH was complicated by PE/E in 22 cases (18%). CONCLUSION: Chronic Hypertension is a prevalent disease, with an elevated incidence in pregnancy, and potential major fetal-maternal complications, obliging medical professionals to a straight and careful follow-up to control BP levels and early diagnose the adversely outcomes.

PP145. Hypertensive disorders of pregnancy: A two year study (2008-2009).

INTRODUCTION: The hypertensive disorders of pregnancy are a leading cause of maternal mortality and morbidity. According to the group studies of NHPBEB 2000 four entities are defined: Chronic Hypertension (CH) previous to pregnancy, Gestational Hypertension (GH), Preeclampsia/Eclampsia (PE/E) and superimposed Preeclampisa/Eclampsia in Chronic Hypertension (PE/E CH). All this entities have different outcomes and require adequate follow-up and specific attitude. OBJECTIVES: Review all cases of hypertensive disorders in a two-year period, its incidences, and related maternal and fetal complications. METHODS: In a retrospective study, from January 2008 to December 2009, all files related with hypertensive disorders, seen in our department, were reviewed. The statistic analysis was based on Excel 2007. RESULTS: The global incidence of hypertensive disorders was 3.8% (309 cases), with each entity with an incidence of: 40% in CH, 40% GH, 25% PE/E and 7% PE/E CH. In terms of demographic characteristics the majority of the population were caucasian (46%) and black (40%), the mean age was of 31years (minimum of 12-maximum of 47), and mainly previous Chronic Hypertension and endocrinologic disorders as co-morbidities (Diabetes Mellitus, obesity and thyroid pathology). The fetal/maternal complications were mainly preterm delivery (26.2%), with a low percentage of Abruptio Placentae (1%). Maternal complications were analysed in terms of ICU admissions of 7%, cardiovascular/renal disorders of 1% and maternal bleeding 1%. No maternal death was described. Fetal outcomes were also studied, specifically in terms of birth weight, with an average of 2794 (500-5480g), apgar index in 1st and 5th minute below seven in respectively, 14% and 3.5%. CONCLUSION: The incidence of maternal complications in our analysis was lower than described in literature. The incidence of preterm delivery was similar to that reported in other studies, mostly due to late pre-terms (>32w).

PP151. Hypertensive complications in pregnancy - Two year study (2008-2009).

INTRODUCTION: Hypertension affects 10% of all pregnancies and accounts for approximately a quarter of all antenatal admissions. Hypertension in pregnancy includes a wide spectrum of conditions, including pre-eclampsia, eclampsia, pre-eclampsia superimposed on chronic hypertension, chronic hypertension, and gestational hypertension. Pregnancies complicated by hypertension are associated with increased risk of adverse fetal, neonatal and maternal outcomes, including preterm birth, fetal growth restriction, perinatal death, acute renal or hepatic failure, ante partum haemorrhage, postpartum haemorrhage and maternal death. Overall pre-eclampsia complicates 5-6% of pregnancies and eclampsia complicates 1-2% of pre-eclamptic pregnancies. OBJECTIVES: Analysis of the maternal complications (incidence of ICU admissions, preeclampsia/eclampsia, renal or cardiovascular acute dysfunction, HELLP syndrome, placental abruption, maternal death) and fetal/neonatal outcome. METHODS: In a retrospective study, from January 2008 to December 2009, all files related with complications of hypertensive disorders, seen in our institution, were analysed. The statistic analysis was based on Excel 2007. RESULTS: Of 309 cases, 123 patients (40%) were found to have gestational hypertension while 121 (40%) suffer chronic hypertension. Ninety patients (29%) have preeclampsia or eclampsia (4 cases). 22 patients with chronic hypertension had a superimposed preeclampsia. Fetal growth restriction, HELLP syndrome and placental abruption were the obstetric complications in 4%, 1% and 1% of the cases, respectively. Additionally, multiple pregnancy and gestational diabetes were noted in 2.6% and 10.7% of the patients. Delivery route was vaginal in 90 patients while 68.9% underwent caesarean section. 6.5% of the patients were admitted to ICU and no woman has died. Preterm delivery occurred in 26.2% of the cases and 2 interruptions of pregnancy before 24weeks were performed due to maternal complications. Intrauterine fetal demise was recorded in 2 cases on admission. CONCLUSION: Women with hypertensive disorders of pregnancy are more likely to have received medical or obstetric interventions such as caesarean section operations. Pregnancies complicated by preeclampsia and eclampsia may be associated with life-threatening complications for both the mother and infant.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture.

PURPOSE: To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. METHODS: Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. RESULTS: Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70% confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80% in membrane type 3, 20% in type 2, and 10% in type 1. CONCLUSION: Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culturey / Avaliação de membranas à base de óleo de polímero de mamona em culturas de células de medula óssea de ratos

PURPOSE: To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. METHODS: Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. RESULTS: Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70 percent confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80 percent in membrane type 3, 20 percent in type 2, and 10 percent in type 1. CONCLUSION: Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

OBJETIVO: Avaliar três formas de cultivo de células-tronco mesenquimais de ratos; e analisar o potencial do polímero de mamona na forma de membrana como arcabouço para CTMs. MÉTODOS: Foram utilizados quatro ratos machos Wistar, de 20 a 30 dias de idade. Aspirados da medula óssea do fêmur e da tíbia foram colhidos com DMEM alta glicose e heparina. As células foram isoladas de três formas diferentes: cultivo direto e com dois tipos de gradientes de densidade. Após 15 dias, foi feita a 1ª passagem e analisada a viabilidade celular com os marcadores Hoerscht 33342 e Iodeto de Propídio. As CTMs foram então caracterizadas por marcadores de superfície, com o auxílio de citômetro de fluxo. Após, três tipos de membrana à base de óleo de polímero de mamona associadas com as CTMS foram mantidas em cultivo por cinco dias, e analisados por microscópio ótico para confirmar o crescimento e a adesão celular. RESULTADOS: Após 15 dias, Os procedimentos que utilizaram gradientes de densidade permitiram o isolamento das CTMs e favoreceram o crescimento celular com a passagem, sendo obtido 70 por cento de confluência após 15 dias em cultura. O procedimento direto não se mostrou eficaz para o isolamento das células. O crescimento das CTMs foi aproximadamente 80 por cento sobre a membrana tipo 3, 20 por cento na tipo 2 e 10 por cento na membrana tipo 1. CONCLUSÃO: Os dois gradientes de concentração Ficoll-Hypaque permitem isolar CTMs de ratos; e especialmente a membrana de polímero de mamona tipo 3 pode ser usada como um bom arcabouço para as CTMs.

Ultrastructure of the hinny (Equus asinus x Equus caballus) seminiferous epithelium.

The gonads and the germinative cells of 3 male hinnies were studied with light and transmission electron microscopy with the aim to observe the development of germ cells and verify the morphological modifications due to the hybridization. The hinny seminiferous epithelium presented Sertoli cells and spermatogonia with normal features and anomalous spermatocytes I. The other cells from the spermatogenic sequence were not seen. Most of the alterations began to occur in the cytes I, which presented nuclear vacuolization and deposits of amorphous material between the carioteca and the nuclear lamina, forming vesicles, or exaggerated chromatin condensation, resulting in pyknosis. In the cytoplasm vacuolization was also observed, besides organelle destruction. The arrest of meiosis due to lock of chromosome homologies leads to germinative cell degeneration and, therefore, the spermatogenesis arrest. This fact causes a profound alteration in the seminiferous epithelium morphology in comparison with the parental species.