Study protocol to investigate biomolecular muscle profile as predictors of long-term urinary incontinence in women with gestational diabetes mellitus.

BACKGROUND: Pelvic floor muscles (PFM) and rectus abdominis muscles (RAM) of pregnant diabetic rats exhibit atrophy, co-localization of fast and slow fibers and an increased collagen type I/III ratio. However, the role of similar PFM or RAM hyperglycemic-related myopathy in women with gestational diabetes mellitus (GDM) remains poorly investigated. This study aims to assess the frequency of pelvic floor muscle disorders and pregnancy-specific urinary incontinence (PS-UI) 12 months after the Cesarean (C) section in women with GDM. Specifically, differences in PFM/RAM hyperglycemic myopathy will be evaluated. METHODS: The Diamater is an ongoing cohort study of four groups of 59 pregnant women each from the Perinatal Diabetes Research Centre (PDRC), Botucatu Medical School (FMB)-UNESP (São Paulo State University), Brazil. Diagnosis of GDM and PS-UI will be made at 24-26 weeks, with a follow-up at 34-38 weeks of gestation. Inclusion in the study will occur at the time of C-section, and patients will be followed at 24-48 h, 6 weeks and 6 and 12 months postpartum. Study groups will be classified as (1) GDM plus PS-UI; (2) GDM without PS-UI; (3) Non-GDM plus PS-UI; and (4) Non-GDM without PS-UI. We will analyze relationships between GDM, PS-UI and hyperglycemic myopathy at 12 months after C-section. The mediator variables to be evaluated include digital palpation, vaginal squeeze pressure, 3D pelvic floor ultrasound, and 3D RAM ultrasound. RAM samples obtained during C-section will be analyzed for ex-vivo contractility, morphological, molecular and OMICS profiles to further characterize the hyperglycemic myopathy. Additional variables to be evaluated include maternal age, socioeconomic status, educational level, ethnicity, body mass index, weight gain during pregnancy, quality of glycemic control and insulin therapy. DISCUSSION: To our knowledge, this will be the first study to provide data on the prevalence of PS-UI and RAM and PFM physical and biomolecular muscle profiles after C-section in mothers with GDM. The longitudinal design allows for the assessment of cause-effect relationships between GDM, PS-UI, and PFMs and RAMs myopathy. The findings may reveal previously undetermined consequences of GDM.

Isolation and characterization of farm pig adipose tissue-derived mesenchymal stromal/stem cells.

Adipose tissue-derived mesenchymal stromal/stem cells (ASCs) are considered important tools in regenerative medicine and are being tested in several clinical studies. Porcine models are frequently used to obtain adipose tissue, due to the abundance of material and because they have immunological and physiological similarities with humans. However, it is essential to understand the effects and safe application of ASCs from pigs (pASCs) as an alternative therapy for diseases. Although minipigs are easy-to-handle animals that require less food and space, acquiring and maintaining them in a bioterium can be costly. Thus, we present a protocol for the isolation and proliferation of ASCs isolated from adipose tissue of farm pigs. Adipose tissue samples were extracted from the abdominal region of the animals. Because the pigs were not raised in a controlled environment, such as a bioterium, it was necessary to carry out rigorous procedures for disinfection. After this procedure, cells were isolated by mechanical dissociation and enzymatic digestion. A proliferation curve was performed and used to calculate the doubling time of the population. The characterization of pASCs was performed by immunophenotyping and cell differentiation in osteogenic and adipogenic lineages. The described method was efficient for the isolation and cultivation of pASCs, maintaining cellular attributes, such as surface antigens and multipotential differentiation during in vitro proliferation. This protocol presents the isolation and cultivation of ASCs from farm pig as an alternative for the isolation and cultivation of ASCs from minipigs, which require strictly controlled maintenance conditions and a more expensive process.

Effect of Bauhinia forficata aqueous extract on the maternal-fetal outcome and oxidative stress biomarkers of streptozotocin-induced diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Bauhinia forficata Link, commonly known as "paw-of-cow", is widely used in Brazilian folk medicine for the treatment of diabetes. AIM OF THIS STUDY: To evaluate the effect of Bauhinia forficata treatment on maternal-fetal outcome and antioxidant systems of streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Virgin female Wistar rats were injected with 40 mg/kg streptozotocin before mating. Oral administration of an aqueous extract of Bauhinia forficata leaves was given to non-diabetic and diabetic pregnant rats at increasing doses: 500 mg/kg from 0 to 4th day of pregnancy, 600 mg/kg from 5th to 14th day and 1000 mg/kg from 15th to 20th day. At day 21 of pregnancy the rats were anaesthetized with ether and a maternal blood sample was collected for the determination superoxide dismutase (SOD) and reduced glutathione (GSH). The gravid uterus was weighed with its contents and fetuses were analyzed. RESULTS AND CONCLUSION: The data showed that the diabetic dams presented an increased glycemic level, resorption, placental weight, placental index, and fetal anomalies, and reduced GSH and SOD determinations, live fetuses, maternal weight gain, gravid uterine weight, and fetal weight. It was also verified that Bauhinia forficata treatment had no hypoglycemic effect, did not improve maternal outcomes in diabetic rats, but it contributed to maintain GSH concentration similarly to non-diabetic groups, suggesting relation with the decreased incidence of visceral anomalies.

Small-for-pregnancy-age rats submitted to exercise: DNA damage in mothers and newborns, measured by the comet assay.

BACKGROUND: Fetal impairment caused by a deleterious intrauterine environment may have long-term consequences, such as oxidative stress and genetic damage. Rats born as small-for-gestational-age (SPA) were submitted to exercise (swimming) before and during pregnancy. The animals exhibited glucose intolerance, reduced general adiposity, and increased maternal and offspring organ weight, showing the benefit of exercise for these rats. We hypothesised that regular exercise in SPA during gestation could prevent DNA damage in these animals and in their offspring, contributing to altered fetal programming of metabolism in the offspring. Severe diabetes was induced by streptozotocin treatment, to obtain SPA newborns. At adulthood, pregnant SPA rats were randomly distributed into two groups: exercised (SPAex - submitted to swimming program) or not-exercised (SPA - sedentary rats). Post-partum, blood was collected for analysis of DNA damage (comet assay) and oxidative stress. SPAex rats presented lower DNA damage levels, decreased lipid peroxidation, and a lower rate of newborns classified as large-for-pregnancy-age. DNA damage was also lower in SPAex newborns. We conclude that swimming applied to SPA pregnant rats contributes to decreased DNA damage and lipid peroxidation in the dams, and decreased DNA damage and macrosomia in their offspring.

Morphological changes in rat rectus abdominis muscle induced by diabetes and pregnancy.

The urethral muscle of diabetic pregnant rats is affected by long-term mild diabetes and short-term severe diabetes, which plays a crucial role in the pathogenesis of pelvic floor disorders. We hypothesized that muscles outside the pelvis are subject to similar changes. The current study aimed at analyzing the effects of long-term mild and short-term severe diabetes on the structure and ultrastructure of fiber muscles and collagen in rats' rectus abdominis (RA) muscle. Therefore, the RA muscle of virgin, pregnant, long-term mild diabetic, short-term severe diabetic, long-term mild diabetic pregnant and short-term severe diabetic pregnant 3-month-old Wistar rats were collected. The structure was analyzed by picrosirius red staining, immunohistochemistry for fast and slow muscle fibers and transmission electron microscopy. We investigated two levels of STZ- induced diabetes: long-term mild diabetes (blood glucose level: 120-200 mg/dL) and short-term severe diabetes (blood glucose level >300 mg/dL). Long-term mild diabetic pregnant and short-term severe diabetic pregnant rats had decreased fast fibers and increased slow fibers, disrupted areas of sarcomere, intermyofibrillar mitochondria and myelin figures in the RA muscle. Both groups enabled us to analyze the specific influence of pregnancy, separately from diabetes. The current study demonstrated that diabetes and pregnancy induced intramuscular transformation and reorganization of RA muscle with a switch of fiber type adjusting their architecture according to intensity and duration of hyperglycemic insult within pregnancy.

Effect of Ginkgo biloba on the reproductive outcome and oxidative stress biomarkers of streptozotocin-induced diabetic rats.

The aim of the present study was to evaluate the effect of Ginkgo biloba treatment (EGb 761, 200 mg kg-1 day-1) administered from day 0 to 20 of pregnancy on maternal reproductive performance and on the maternal and fetal liver antioxidant systems of streptozotocin-induced diabetic Wistar rats. On day 21 of pregnancy, the adult rats (weighing approximately 250 +/- 50 g, minimum number = 13/group) were anesthetized to obtain maternal and fetal liver samples for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and total glutathione (GSH-t) determinations. The uterus was weighed with its contents. The diabetic (G3) and treated diabetic (G4) groups of rats presented significant maternal hyperglycemia, reduced term pregnancy rate, impaired maternal reproductive outcome and fetal-placental development, decreased GSH-Px (G3 = G4 = 0.6 +/- 0.2) and SOD (G3 = 223.0 +/- 84.7; G4 = 146.1 +/- 40.8), and decreased fetal CAT activity (G3 = 22.4 +/- 10.6; G4 = 34.4 +/- 14.1) and GSH-t (G3 = G4 = 0.3 +/- 0.2), compared to the non-diabetic groups (G1, untreated control; G2, treated). For G1, maternal GSH-Px = 0.9 +/- 0.2 and SOD = 274.1 +/- 80.3; fetal CAT = 92.6 +/- 82.7 and GSH-t = 0.6 +/- 0.5. For G2, G. biloba treatment caused no toxicity and did not modify maternal or fetal-placental data. EGb 761 at the nontoxic dose used (200 mg kg-1 day-1), failed to modify the diabetes-associated increase in maternal glycemia, decrease in pregnancy rate, decrease in antioxidant enzymes, and impaired fetal development when the rats were treated throughout pregnancy (21 days).

Isolation and characterization of farm pig adipose tissue-derived mesenchymal stromal/stem cells

Adipose tissue-derived mesenchymal stromal/stem cells (ASCs) are considered important tools in regenerative medicine and are being tested in several clinical studies. Porcine models are frequently used to obtain adipose tissue, due to the abundance of material and because they have immunological and physiological similarities with humans. However, it is essential to understand the effects and safe application of ASCs from pigs (pASCs) as an alternative therapy for diseases. Although minipigs are easy-to-handle animals that require less food and space, acquiring and maintaining them in a bioterium can be costly. Thus, we present a protocol for the isolation and proliferation of ASCs isolated from adipose tissue of farm pigs. Adipose tissue samples were extracted from the abdominal region of the animals. Because the pigs were not raised in a controlled environment, such as a bioterium, it was necessary to carry out rigorous procedures for disinfection. After this procedure, cells were isolated by mechanical dissociation and enzymatic digestion. A proliferation curve was performed and used to calculate the doubling time of the population. The characterization of pASCs was performed by immunophenotyping and cell differentiation in osteogenic and adipogenic lineages. The described method was efficient for the isolation and cultivation of pASCs, maintaining cellular attributes, such as surface antigens and multipotential differentiation during in vitro proliferation. This protocol presents the isolation and cultivation of ASCs from farm pig as an alternative for the isolation and cultivation of ASCs from minipigs, which require strictly controlled maintenance conditions and a more expensive process.

Intrauterine Growth Restricted Rats Exercised before and during Pregnancy: Maternal and Perinatal Repercussions.

This study aimed at evaluating the effect of swimming before and during pregnancy on rats born with intrauterine growth restriction (IUGR) and their offspring. For this, nondiabetic and streptozotocin-induced severely diabetic (SD) pregnant rats were mated and generated offspring with appropriate (control, C) and small (IUGR) for pregnancy age, respectively. Following that, C and IUGR groups were further distributed into nonexercised control (C), exercised control (Cex), nonexercised IUGR (IUGR), and exercised IUGR (IUGRex). IUGR rats presented lower mating rate than control rats. Regardless of physical exercise IUGR rats presented decreased body weight from birth to lactation. At 90 days of life, IUGR rats presented glucose intolerance. Maternal organ weights were increased and relative adiposity of IUGRex rats was lower than Cex. IUGR and IUGRex offspring presented reduced body weight than C and Cex, respectively. IUGRex dams presented an increased rate of appropriate for pregnancy age newborns. IUGEex male and female offspring relative brain weight was increased compared with Cex. Therefore, swimming before and during pregnancy prevented glucose intolerance, reduced general adiposity, and increased maternal and offspring organ weight in rats, showing the benefit of physical exercise for IUGR rats.

Intrauterine Growth Restricted Rats Exercised at Pregnancy: Maternal-Fetal Repercussions.

To evaluate the effect of swimming in pregnant rats born with intrauterine growth restriction (IUGR) and their offspring, IUGR rats were obtained using the streptozotocin-induced severe diabetic (SD) rats. In this study, the nondiabetic parental generation presented 10 rats and diabetic parental generation presented 116 rats. Of these, the mated nondiabetic female rats were 10 and the number of diabetic rats was 45. In relation to term pregnancy, there were 10 animals in the nondiabetic group and 15 rats in the diabetic group. In the offspring of SD rats (IUGR group), 43 females were classified as small for pregnancy age, 19 rats were classified as appropriate for pregnancy age, and 0 female was classified as large for pregnancy age. The nondiabetic and SD pregnant rats generated offspring with appropriate (control [C]) and small (IUGR) weight for pregnancy age, respectively. At adult life, the C group was maintained as nonexercised C group and IUGR rats were distributed into 2 subgroups, namely, nonexercised (IUGR) and exercised (IUGRex). The rate of mated rats in the IUGR group was reduced compared to the C group. During pregnancy, the IUGR rats presented hyperinsulinemia, impaired reproductive outcomes, decreased body weight, hypertriglyceridemia, and hyperlactacidemia. The IUGRex presented reduced insulin and triglyceride levels. Thus, swimming improved lipid metabolism and increased insulin sensitivity. However, the offspring showed retarded growth, reinforcing the need to stimulate the exercise practice in women under supervision with different professional expertise to promote appropriate gestational conditions and improve perinatal outcomes.

Pancreatic beta-cell defects in women at risk of type 2 diabetes.

We evaluated insulin release and insulin sensitivity in women with basal and/or postprandial hyperglycemia but normal oral glucose tolerance test (OGTT) in previous pregnancy (GHG). These women were individually matched with females without previous hyperglycemia (NGT). Both groups consisted of normal glucose-tolerant women at the time of this study. They underwent OGTT (75 g; n=32 pairs) and hyperglycemic clamp experiments (10 mmoll(-1); n=27 pairs) with plasma glucose, insulin, and C-peptide measurements and calculation of insulinogenic index, first- and second-phase insulin release, and insulin sensitivity index (ISI). The GHG group showed higher glycosylated hemoglobin levels (6.2+/-0.6% versus 5.8+/-0.8%; P<0.05); lower insulinogenic index at 30 min (134.03+/-62.69 pmol mmol(-1) versus 181.59+/-70.26 pmol mmoll(-1); P<0.05) and diminished C-peptide response in relation to glucose (4.05+/-0.36 nmol mmol(-1) versus 4.23+/-0.36 nmol mmol(-1); P<0.05) at OGTT. Both groups did not show difference in insulin secretion and ISI by hyperglycemic clamp technique. We concluded that in up to 12 years from index pregnancy, women with previous GHG, presenting normal glucose tolerance and well-matched with their controls, showed beta-cell dysfunction without change in ISI. As women with previous GHG are at risk of type 2 diabetes, beta-cell dysfunction may be its primary defect.

The oral glucose tolerance test is a poor predictor of hyperglycemia during pregnancy.

1. In order to assess the efficacy of the use of the diurnal plasma glucose profile rather than that of the glucose tolerance test (GTT) to predict hyperglycemia during pregnancy, we compared the results of the two tests. A total of 192 pregnant women seen at the Prenatal Clinic of the Faculty of Medicine of Botucatu were submitted to the glucose tolerance test (GTT) and determination of diurnal plasma glucose profile. 2. On the basis of two blood tests (GTT and diurnal plasma glucose profile), the subjects were divided into four groups: Group I-A, normal GTT and profile (79 patients, 41.2%); Group I-B, normal GTT and altered profile (63 patients, 32.8%); Group II-A, altered GTT and normal profile (18 patients, 9.4%); Group II-B, altered GTT and profile (32 patients, 16.7%). 3. Large babies were delivered by 25.6% of Group I-A, 53.8% of Group I-B, 28.6% of Group II-A and 51.9% of Group II-B patients. Group I-A patients are normoglycemic, Group I-B patients have intolerance to carbohydrates, protein and lipids, Group II-A patients have intolerance to high carbohydrate amounts, especially in the form of glucose, and Group II-B patients are diabetic. 4. We propose that Group I-A patients should receive no treatment, Group II-A patients should be advised to avoid excess carbohydrate intake and Groups I-B and II-B patients should be placed on a low-calorie diet and treated with insulin if necessary to obtain normal blood glucose levels. 5. Routine determination of blood glucose levels under fasting conditions represents a screening method for diabetes and values of greater than or equal to 90 mg/dl identify a population at risk that should be submitted to GTT and determination of plasma glucose profile.

Diabetes and experimental pregnancy in rats: course of maternal blood glucose levels and its repercussions on the blood glucose levels and pancreas of newborn pups.

The objective of the present investigation was to determine the course of maternal blood glucose levels in pregnant rats and its repercussions on the glucose levels and pancreas of their newborn pups. Diabetes was induced by alloxan (42 mg/kg body weight) and streptozotocin (40 mg/kg). Sixty-two pregnant Wistar rats weighing 180 to 250 g were divided into a control group and two groups with moderate (120 to 200 mg/dl glucose) and severe diabetes (greater than 200 mg/dl glucose), respectively. Blood glucose levels were measured in the dams on the 1st, 14th, and 21st days of pregnancy and in the pups at birth. The results were pooled for each litter. The fetal pancreases were removed after cesarian section performed on the 21st day of pregnancy, pooled for each litter and processed for histopathologic examination by light microscopy. Maternal blood glucose levels were significantly increased compared with the first day of pregnancy in both normal and diabetic rats starting on the 14th day of pregnancy. Fetal blood glucose levels correlated with maternal levels. The histopathologic changes characterized by vacuolization and basophilia of the cytoplasm of endocrine pancreas of newborn pups from dams with moderate or severe diabetes suggested pancreatic hyperactivity.

Studies of natural killer cells in pregnancy-induced hypertension.

The number and activity of natural killer (NK) cells were studied in 20 patients with pregnancy-induced hypertension (PIH), 15 uncomplicated pregnant women and 16 healthy non-pregnant women. All the pregnant women were primigravidae and were evaluated during the third trimester of gestation. Peripheral blood NK cells were detected with monoclonal antibodies by indirect immunofluorescence and cytotoxic activity was measured using a single-cell assay against K562 target cells. Hypertensive pregnant women had an increased number of circulating NK cells associated with a significant decrease of NK activity. The cytotoxic activity was significantly lower in normal pregnant and PIH women when compared with non-pregnant controls. The onset of immature NK cells in peripheral blood and the impairment of their cytotoxic activity in PIH patients may be associated with hormones and immunosuppressive substances produced by tissues occurring at the maternal-fetal interface.

Effects of hypertension on maternal adaptations to pregnancy: experimental study on spontaneously hypertensive rats.

CONTEXT: Animal models for essential hypertension have been used for understanding the human pathological conditions observed in pregnant hypertensive women. OBJECTIVE: To study the possible effects of pregnancy on hypertension and of hypertension on pregnancy in spontaneously hypertensive rats (SHR), and in their normotensive Wistar-Kyoto (WKY) counterparts. TYPE OF STUDY: Comparative study using laboratory animals. SETTING: Animal Research Laboratory of Clinical Medicine at the Medical School of Botucatu, São Paulo State University, Brazil. SAMPLE: Ten to twelve-week-old virgin female normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The animals were separated into four groups: 15 pregnant spontaneously hypertensive rats (SHR-P), 10 non-pregnant spontaneously hypertensive rats (SHR-NP), 15 pregnant normotensive rats (WKY-P), and 10 non-pregnant normotensive rats (WKY-NP). MAIN MEASUREMENTS: The blood pressure was evaluated by the tail cuff method, in rats either with or without prior training for the handling necessary for tail cuff measurements. The maternal volume expansion was indirectly evaluated by weight gain, and by systemic parameters as hematocrit, hemoglobin, total protein, albumin and sodium retention. The perinatal outcome of pregnancy was evaluated by analysis of resorptions, litter size, rate of low weight and number of stillbirths. RESULTS: The late fall in blood pressure in the pregnant SHR strain and in the normotensive WKY strain can only be detected in rats previously trained to accept the handling necessary for the tail cuff measurement. During pregnancy the body weight gain was significantly higher in WKY than in SHR rats. Systemic parameters were significantly lower in pregnant WKY rats than in non-pregnant WKY rats, while no differences were observed between pregnant and non-pregnant SHR groups. In pregnant WKY rats the sodium retention was higher from the 13th day onwards, while in SHR rats this occurred only on the 21st day. The characteristics of reproductive function such as number and weight of fetus, perinatal mortality and the resorption rate were significantly affected in the SHR strain. CONCLUSION: The SHR strain may be considered as a model for chronic hypovolemic maternal hypertension, with the fetal growth retardation being determined by this hypovolemic state.

Study of the evolution of the placenta and fetal pancreas in the pathophysiology of growth retardation intrauterine due to restricted maternal diet.

CONTEXT: Intrauterine growth retard (IUGR) continues to be a significant perinatology problem at the end of this century. The nature of the etiologic agent, the time when the attack occurred during pregnancy and its duration affect the type of IUGR. OBJECTIVE: To study the evolution of fetal pancreas and placenta between the 18th and 21st day of pregnancy in rats submitted to maternal protein-calorie restriction. DESIGN: Randomized controlled trial on laboratory animal. SAMPLE: Forty-one normoglycemic pregnant Wistar rats. INTERVENTION: Rats were divided into six experimental groups according to their access to food and date of cesarean section (18th or 21st day): control with free access to food; diet restricted to 25% introduced on 1st day of pregnancy; and diet restricted to 25% after the 3rd day of pregnancy. MAIN MEASUREMENTS: Newborn weight, placenta weight, histopathological study (morphological histochemistry). RESULTS: Maternal protein-calorie malnutrition caused intrauterine growth retard (IUGR) after the 18th day of pregnancy. Dietary restriction did not interfere with the morphology of the fetal pancreas and the immunohistochemical study of the placenta showed that glycogen stores were decreased between the 18th and 21st day in the control group and in a diet restricted to 25% from the first day of pregnancy. Dietary restriction after the 3rd day of pregnancy led to low placental glycogen concentrations on the 18th day and disappearance on the 21st day. CONCLUSION: The pathophysiology of IUGR due to maternal protein-calorie restriction in rats is related to lower placental weight and low placental glycogen stores.

[Locally advanced cancer of the breast (LACB) and identification of estrogen receptors with monoclonal antibodies]. / Considerações sobre o câncer localmente avançado de mama (CLAM) acrescidas de identificação dos receptores de estrogênios usando anticorpos monoclonais.

The effects of therapy in locally advanced breast cancer submitted to combined conventional telecobalt therapy plus chemotherapy with cyclophosphamide and 5-fluorouracil were studied in 49 patients. Associated to radical mastectomy in operable cases. Local tumor control was achieved in 86.7%. There were no local recurrences in those submitted to surgery but they reached 21.7% in inoperable patients who received only radiation therapy and chemotherapy. The median follow-up time for dead patients was 29.5 months and for living patients 79.3 months. The index of complete responses was 24.5% and the median disease free interval was 22.9 months. The overall survival rate, between three and five years, was 32.7%. Estrogen receptors were identified by using immunohistochemical assay ER-ICA and monoclonal antibody H222-SP gamma, Abbott. There were no differences in the complete response index, disease free interval and survival rates, among ER-positive and ER-negative patients, explained by the far advanced stage of the disease. ER-positivity was significantly correlated with histological features of the tumors: cell differentiation, presence of elastosis, absence of lymphocytic infiltration and absence of tumor necrosis.

Streptozotocin-induced diabetes models: pathophysiological mechanisms and fetal outcomes.

Glucose homeostasis is controlled by endocrine pancreatic cells, and any pancreatic disturbance can result in diabetes. Because 8% to 12% of diabetic pregnant women present with malformed fetuses, there is great interest in understanding the etiology, pathophysiological mechanisms, and treatment of gestational diabetes. Hyperglycemia enhances the production of reactive oxygen species, leading to oxidative stress, which is involved in diabetic teratogenesis. It has also been suggested that maternal diabetes alters embryonic gene expression, which might cause malformations. Due to ethical issues involving human studies that sometimes have invasive aspects and the multiplicity of uncontrolled variables that can alter the uterine environment during clinical studies, it is necessary to use animal models to better understand diabetic pathophysiology. This review aimed to gather information about pathophysiological mechanisms and fetal outcomes in streptozotocin-induced diabetic rats. To understand the pathophysiological mechanisms and factors involved in diabetes, the use of pancreatic regeneration studies is increasing in an attempt to understand the behavior of pancreatic beta cells. In addition, these studies suggest a new preventive concept as a treatment basis for diabetes, introducing therapeutic efforts to minimize or prevent diabetes-induced oxidative stress, DNA damage, and teratogenesis.

Mild diabetes models and their maternal-fetal repercussions.

The presence of diabetes in pregnancy leads to hormonal and metabolic changes making inappropriate intrauterine environment, favoring the onset of maternal and fetal complications. Human studies that explore mechanisms responsible for changes caused by diabetes are limited not only for ethical reasons but also by the many uncontrollable variables. Thus, there is a need to develop appropriate experimental models. The diabetes induced in laboratory animals can be performed by different methods depending on dose, route of administration, and the strain and age of animal used. Many of these studies are carried out in neonatal period or during pregnancy, but the results presented are controversial. So this paper, addresses the review about the different models of mild diabetes induction using streptozotocin in pregnant rats and their repercussions on the maternal and fetal organisms to propose an adequate model for each approached issue.

Genotoxicity evaluation in severe or mild diabetic pregnancy in laboratory animals.

This study aimed to evaluate the genotoxicity (DNA damage levels) in lymphocyte samples from pregnant Wistar rats with severe or mild diabetes and in whole blood samples from their newborns. Wistar female rats (1 and 90 days of age) and male rats (approximately 90 days of age) were used. The experiment consisted of 2 experimental groups (n=8 animals/group): 1) rats with severe diabetes, 2) rats with mild diabetes. For mild diabetes induction, the rats received streptozotocin (STZ) subcutaneously (100 mg/kg body weight) at day of birth, and those showing glycemia from 120 to 300 mg/dL in their adult life were included. For induction of severe diabetes, adult rats received 40 mg/kg STZ (intravenous route), and those showing glycemia > 300 mg/dL were included. At day 21 of pregnancy, the rats were anesthetized and euthanized for removal of maternal and fetal blood samples for determination of the oxidative DNA damage by applying Endo III and Fpg using the comet assay. Thus, the rats with mild diabetes and their offspring showed higher Fpg-sensitive sites, reflecting the damage resulting from hyperglycemia. The rats with severe diabetes and their offspring showed higher oxidative DNA damage detected by Fpg and Endo III-sensitive sites, showing general repercussions related to diabetes. The enzymatic treatment for DNA damage evidenced that the maternal repercussions of diabetes are associated with oxidative DNA damage of their newborn, which was not reflected using only the analysis of DNA damage free of the enzymes.

PP061. The role of heat shock protein 60 and 70 in early- and late-onset preeclampsia differentiation.

INTRODUCTION: Preeclampsia is a human pregnancy-specific syndrome characterized by the onset of hypertension and proteinuria. These manifestations may occur before the 34th week of gestation or from this period on, being denominated early-onset or late-onset preeclampsia respectively. The etiology of both disorders seems to differ qualitatively; therefore, different strategies of prevention and treatment must be studied. OBJECTIVES: The aim of the present study is to determine whether the plasma levels of heat-shock proteins Hsp60 and Hsp70 as well as specific antibodies anti-Hsp60 and anti-Hsp70 may differentiate early-onset from late-onset preeclampsia. METHODS: We evaluated 175 pregnant women with PE (55 early-onset PE and 120 late-onset PE). Plasma was obtained from peripheral blood and Hsp60, Hsp70 as well as anti-Hsp60 and anti-Hsp70 antibody levels were determined by enzyme immunoassay. Uric acid levels were also determined in the plasma of patients. For statistical analyses, the Mann-Whitney U-test and the Spearman rank order correlation were applied with significance level set at 5%. RESULTS: Hsp70 levels obtained from early-onset PE group were significantly higher than the late-onset PE women and showed positive correlation with uric acid (r=0.4547; p=0.0028). The Hsp60 production was similar in both groups. Our results also indicate that there was no significant difference of anti-Hsp60 and anti-Hsp70 antibody levels between women with early- and late-onset PE. However,these antibody levels were high,indicating a strong relationship with the production of HSP60 and Hsp70 protein. CONCLUSION: Association between levels of Hsp70 and uric acid in plasma of patients with early-onset PE seems to reflect the oxidative stress in this group of patients. This study provides evidence that Hsp70 determination may be utilized to assess the differentiation between early- and late-onset PE. FINANCIAL SUPPORT: FAPESP 2010/09241-2.