Micronutrient prenatal supplementation prevents the development of hypertension and vascular endothelial damage induced by intrauterine malnutrition

Aims: The premise that intrauterine malnutrition plays an important role in the development of cardiovascular and renal diseases implies that these disorders can be programmed during fetal life. Here, we analyzed the hypothesis that supplementation with mixed antioxidant vitamins and essential mineral in early life could prevent later elevation of blood pressure and vascular and renal dysfunction associated with intrauterine malnutrition. Main methods: For this, female Wistar rats were randomly divided into three groups on day 1 of pregnancy: control fed standard chow ad libitum; restricted group fed 50% of the ad libitum intake and a restricted plus micronutrient cocktail group treated daily with a combination of micronutrient (selenium, folate, vitamin C and vitamin E) by oral gavage. Key findings: In adult offspring, renal function and glomerular number were impaired by intrauterine malnutrition. and the prenatal micronutrient treatment did not prevent it. However, increased blood pressure and reduced endothelium-dependent vasodilation were prevented by the micronutrient prenatal treatment. Intrauterine malnutrition also led to reduced NO production associated with increased superoxide generation, and these parameters were fully normalized by this prenatal treatment. Significance: Our current findings indicate that programming alterations during fetal life can be prevented by interventions during the prenatal period, and that disturbance in availability of both antioxidant vitamins and mineral may play a crucial role in determining the occurrence of long-term cardiovascular injury. (C) 2009 Elsevier Inc. All rights reserved.

Environmental modulation of ethanol-induced locomotor activity: Correlation with neuronal activity in distinct brain regions of adolescent and adult Swiss mice

Drug abuse is a concerning health problem in adults and has been recognized as a major problem in adolescents. induction of immediate-early genes (IEG), such as c-Fos or Egr-1, is used to identify brain areas that become activated in response to various stimuli, including addictive drugs. It is known that the environment can alter the response to drugs of abuse. Accordingly, environmental cues may trigger drug-seeking behavior when the drug is repeatedly administered in a given environment. The goal of this study was first to examine for age differences in context-dependent sensitization and then evaluate IEG expression in different brain regions. For this, groups of mice received i.p. ethanol (2.0 g/kg) or saline in the test apparatus, while other groups received the solutions in the home cage, for 15 days. One week after this treatment phase, mice were challenged with ethanol injection. Acutely, ethanol increased both locomotor activity and IEG expression in different brain regions, indistinctly, in adolescent and adult mice. However, adults exhibited a typical context-dependent behavioral sensitization following repeated ethanol treatment, while adolescent mice presented gradually smaller locomotion across treatment, when ethanol was administered in a paired regimen with environment. Conversely, ethanol-treated adolescents expressed context-independent behavioral sensitization. Overall, repeated ethanol administration desensitized IEG expression in both adolescent and adult mice, but this effect was greatest in the nucleus accumbens and prefrontal cortex of adolescents treated in the context-dependent paradigm. These results suggest developmental differences in the sensitivity to the conditioned and unconditioned locomotor effects of ethanol. (C) 2008 Elsevier B.V. All rights reserved.

Influence of age on the development of immunological lung response in intrauterine undernourishment

Objective: We investigated the effect of intrauterine undernourishment on some features of asthma using a model of allergic lung inflammation in rats. The effects of age at which the rats were challenged (5 and 9 wk) were also evaluated. Methods: Intrauterine undernourished offspring were obtained from dams that were fed 50% of the nourished diet of counterparts and were immunized at 5 and 9 wk of age. They were tested for immunoglobulin E anti-ova titers (by passive cutaneous anaphylaxis), cell count in the bronchoal-veolar fluid, leukotriene concentration, airway reactivity, mucus production, and blood corticosterone and leptin concentrations 21 d, after immunologic challenge. Results: Intrauterine undernourishment significantly reduced the antigen-specific immunoglobulin E production, inflammatory cell infiltration into airways, mucus secretion, and production of leukotrienes B-4/C-4 in the lungs in both age groups compared with respective nourished rats. The increased reactivity to methacholine that follows antigen challenge was not affected by intrauterine undernourishment. Corticosterone levels increased with age in the undernourished rats` offspring, but not in the nourished rats` offspring. Undernourished offspring already presented high levels of corticosterone before inflammatory stimulus and were not modified by antigen challenge. Leptin levels increased with challenge in the nourished rats but not in the undernourished rats and could not be related to corticosterone levels in the. undernourished rats. Conclusion: Intrauterine undernourishment has a striking and age-dependent effect on the off spring, reducing lung allergic inflammation. (C) 2008 Elsevier Inc. All rights reserved.

Morfologia e funcionalidade do pneumócito tipo II e sua relação e variação com a idade gestacional em bovinos

Este estudo objetivou caracterizar a presença de pneumócitos tipo II e o início da produção de lipoproteína surfactante em bovinos, correlacionando a idade gestacional com a síntese de surfactante durante o desenvolvimento fetal. Pulmões de fetos com quatro meses de idade gestacional estavam na fase canalicular de desenvolvimento, sem a presença de pneumócitos tipo II ou bandas eletroforéticas compatíveis com a presença de proteínas surfactante. No 5° mês gestacional, os pulmões dos fetos encontravam-se em fase de saculação terminal, com a presença de alvéolos por epitélio cúbico, com áreas formadas por pneumócitos I e II. Nesse período ainda não foi possível identificar proteína surfactante nos pulmões. Esses órgãos em fetos com seis meses de idade gestacional estavam em fase de saco terminal, com presença de pneumócitos tipo I e II. Nessa fase a análise para determinação protéica do surfactante de feto bovino (SDS - PAGE) demonstrou presença de bandas entre 26 e 36kDa, confirmando produção de SP - A, proteína surfactante encontrada em maior quantidade. A partir do 7° mês gestacional, a fase de saco terminal é mais evidente e complexa, com desenvolvimento de intensa vascularização. O pneumócito tipo I apresentava aspecto mais pavimentoso, e o tipo II apresentava aspecto mais globoso. Na análise SDS - PAGE do lavado bronco - alveolar, bandas de proteína surfactante com aspecto similar ao de animais recém-nascidos foram encontradas. Em recém-nascidos, pulmões na fase alveolar foram observados com pneumócitos tipo I e II característicos. O perfil das bandas do lavado bronco-alveolar dos recém-nascidos foi igual ao de animais adultos. Esses achados sugerem que um animal nascido precocemente, a partir dos sete meses de gestação, teria sua sobrevivência garantida devido a uma possível funcionalidade do sistema respiratório do feto, pois o pulmão possuiria as características necessárias para a síntese de proteínas surfactantes. Entretanto, mais estudos clínicos sobre a funcionalidade do sistema respiratório abrem novas fronteiras de experimentos sobre fisiologia respiratória em recém-nascidos bovinos.

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Melatonin (MEL) acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM) on bovine embryos. This study tested the addition of MEL to maturation medium (MM) with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS) at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL) or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.

Efeito do número da passagem e do gênero das células doadoras de núcleo no desenvolvimento de bovinos produzidos por transferência nuclear

Objetivou-se com este trabalho avaliar o efeito do número da passagem e do sexo das células doadoras de núcleo no desenvolvimento embrionário e fetal após transferência nuclear. Para isso, oócitos bovinos foram maturados, enucleados e reconstruídos com células somáticas de animal adulto. Após a fusão e ativação química, os zigotos reconstituídos foram cultivados em Charles Rosenkranz 2 (CR2) com monocamada de células da granulosa a 38,8ºC em atmosfera umidificada a 5% de CO2 em ar, durante sete dias, e transferidos para receptoras sincronizadas. As taxas de clivagem e desenvolvimento a blastocisto de embriões reconstruídos com células cultivadas por tempo maior foram inferiores às obtidas com os demais tempos de cultivo. Além disso, os blastocistos produzidos não resultaram no desenvolvimento de uma gestação a termo. Embora a taxa de clivagem em embriões fêmeas tenha sido maior, o número de embriões que atingiram o estádio de blastocisto foi maior nos embriões machos. No período gestacional, fêmeas apresentaram maior taxa de aborto entre 90 e 120 dias de gestação. Esses resultados indicam que células doadoras de núcleos cultivados por longos períodos dificultam a produção de blastocistos e aumentam as chances de perdas durante a gestação. Embriões clonados machos têm maior competência para se desenvolver a blastocisto e resultam em menor taxa de perda gestacional.

Sensitivity to halothane and its relationship to the development of PSE (Pale, Soft, Exudative) meat in female lineage broilers

This work aimed to evaluate female lineage broilers for halothane sensitivity and for their susceptibility to the subsequent development of PSE meat. The halothane test was carried out in an anesthetic chamber with 3.0% halothane. The unconscious birds were examined for leg muscle rigidity. If one or both legs became extended and rigid, the birds were classified as halothane sensitive (HAL+), while unresponsive birds were classified as halothane negative (HAL-). The results showed that of 298 birds aged 42 days old, 95.6% were HAL- and 4.4% were HAL+. A sample of pectoralis major muscle was collected from HAL- (n=105) and HAL+ (n=13) birds. The pH and breast fillet color were determined at 4ºC, 24 hours post-mortem. Interestingly, only 2.5% of HAL+ birds displayed PSE meat characteristics compared to 12.7% of HAL- individuals. The halothane test demonstrated that female lineage broilers displayed very little sensitivity towards halothane, indicating that the development of PSE meat is related to other environmental factors.

Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.

Long-term social recognition memory in adult male rats: factor analysis of the social and non-social behaviors

A modified version of the intruder-resident paradigm was used to investigate if social recognition memory lasts at least 24 h. One hundred and forty-six adult male Wistar rats were used. Independent groups of rats were exposed to an intruder for 0.083, 0.5, 2, 24, or 168 h and tested 24 h after the first encounter with the familiar or a different conspecific. Factor analysis was employed to identify associations between behaviors and treatments. Resident rats exhibited a 24-h social recognition memory, as indicated by a 3- to 5-fold decrease in social behaviors in the second encounter with the same conspecific compared to those observed for a different conspecific, when the duration of the first encounter was 2 h or longer. It was possible to distinguish between two different categories of social behaviors and their expression depended on the duration of the first encounter. Sniffing the anogenital area (49.9% of the social behaviors), sniffing the body (17.9%), sniffing the head (3%), and following the conspecific (3.1%), exhibited mostly by resident rats, characterized social investigation and revealed long-term social recognition memory. However, dominance (23.8%) and mild aggression (2.3%), exhibited by both resident and intruders, characterized social agonistic behaviors and were not affected by memory. Differently, sniffing the environment (76.8% of the non-social behaviors) and rearing (14.3%), both exhibited mostly by adult intruder rats, characterized non-social behaviors. Together, these results show that social recognition memory in rats may last at least 24 h after a 2-h or longer exposure to the conspecific.

Reproductive behavior, development and eye regression in the cave armored catfish, Ancistrus cryptophthalmus Reis, 1987 (Siluriformes: Loricariidae), breed in laboratory

The troglobitic armored catfish, Ancistrus cryptophthalmus (Loricariidae, Ancistrinae) is known from four caves in the São Domingos karst area, upper rio Tocantins basin, Central Brazil. These populations differ in general body shape and degree of reduction of eyes and of pigmentation. The small Passa Três population (around 1,000 individuals) presents the most reduced eyes, which are not externally visible in adults. A small group of Passa Três catfish, one male and three females, reproduced spontaneously thrice in laboratory, at the end of summertime in 2000, 2003 and 2004. Herein we describe the reproductive behavior during the 2003 event, as well as the early development of the 2003 and 2004 offsprings, with focus on body growth and ontogenetic regression of eyes. The parental care by the male, which includes defense of the rock shelter where the egg clutch is laid, cleaning and oxygenation of eggs, is typical of many loricariids. On the other hand, the slow development, including delayed eye degeneration, low body growth rates and high estimated longevity (15 years or more) are characteristic of precocial, or K-selected, life cycles. In the absence of comparable data for close epigean relatives (Ancistrus spp.), it is not possible to establish whether these features are an autapomorphic specialization of the troglobitic A. cryptophthalmus or a plesiomorphic trait already present in the epigean ancestor, possibly favoring the adoption of the life in the food-poor cave environment. We briefly discuss the current hypotheses on eye regression in troglobitic vertebrates.

Histomorfometria das meninges encefálicas de ratos Wistar em diferentes faixas etárias

O desenvolvimento do sistema nervoso é bastante complexo, existindo poucos estudos sobre a organização dos envoltórios cerebrais relacionados ao crescimento encefálico. Utilizando como modelo experimental o rato, analisaram-se os diferentes aspectos estruturais e morfométricos da paquimeninge e leptomeninge durante o processo de envelhecimento. Foram utilizados quatro grupos de ratos em diferentes faixas etárias e analisadas as meninges em microscopias de luz e eletrônica. Verificamos que o grupo de ratos adultos apresentou uma maior área de fibras colágenas tanto do tipo I e quanto do tipo III, em relação aos outros grupos. Encontramos também que as fibras colágenas do tipo III em todos os grupos analisados ocupam uma maior área quando comparados com as fibras do tipo I. Os resultados revelam que a coloração de Weigert Oxona, que mostra fibras elásticas, elaunínicas e oxitalânicas, apresentou uma diferença estatisticamente maior de fibras quando comparados com as colorações de Weigert e Verhoeff, que mostra fíbras elaunínicas e elásticas, respectivamente. Os resultados ultra-estruturais demonstraram a presença de muitos fibroblastos e mitocôndrias tanto na paquimeninge como nas leptomeninges dos grupos de ratos neonatos e adultos, indicativo de alta atividade celular e conseqüentemente, intensa formação de tecido conjuntivo. Como as fibras colágenas do tipo III atuam na manutenção da estrutura de tecidos delicados e expansíveis, o estudo mostra que as funções das meninges encefálicas não estão relacionadas apenas com a resistência a trações e tensões a que estão sujeitas o encéfalo. Mas também a função relacionada com a distensibilidade dos vasos meníngeos e cerebrais de acordo com a necessidade do aporte sanguíneo em diversas funções específicas regionais do tecido nervoso.

Pode a atividade física materna modular a programação fetal induzida pela nutrição?

Existe considerável evidência para a indução de diferentes fenótipos em reposta às variações no ambiente fetal e neonatal. O aporte inadequado de nutrientes no período crítico do desenvolvimento está associado ao risco alto de doenças metabólicas na vida adulta, este fenômeno biológico é chamado de programação. A atividade física durante a gestação resulta em adaptações fisiológicas da mãe e no aumento da disponibilidade de nutrientes e oxigênio no espaço feto-placentário. Este trabalho tem como objetivo discutir os mecanismos da indução de programação fetal pela nutrição e o provável efeito modulador da atividade física durante a gestação. Foram utilizadas as bases de dados do Medline Pubmed, Lilacs e Bireme, com publicações entre 1990 até 2008. Os termos de indexação utilizados foram: nutrition, fetal programming, gestation, physical activity, physical exercise, metabolism. Em conclusão, o aporte inadequado de nutrientes programa o aparecimento de doenças metabólicas na vida adulta, enquanto que a atividade física durante a gestação aumenta a disponibilidade de nutrientes e oxigênio, repercutindo positivamente no crescimento fetal e no peso ao nascer.

Reduced cortical renal GLUT1 expression induced by angiotensin-converting enzyme inhibition in diabetic spontaneously hypertensive rats

Diabetes in spontaneously hypertensive rats is associated with cortical renal GLUT1 and GLUT2 overexpression. Our objective was to evaluate the effect of the angiotensin-converting enzyme blockade on cortical renal GLUT1 and GLUT2 expression, urinary albumin and urinary TGF-β1. Streptozotocin, 50 mg/kg, or citrate buffer (N = 16) was administered as a single injection into the tail vein in adult spontaneously hypertensive rats (~260 g). Thirty days later, these diabetic spontaneously hypertensive rats received ramipril by gavage: 0.01 mg·kg-1·day-1 (D0.01, N = 14), 1 mg·kg-1·day-1 (D1, N = 9) or water (D, N = 11) for 15 days. Albumin and TGF-β1 (24-h urine), direct arterial pressure, renal tissue angiotensin-converting enzyme activity (fluorometric assay), and GLUT1 and GLUT2 protein levels (Western blot, renal cortex) were determined. Glycemia and glycosuria were higher (P < 0.05) in the diabetic rats compared with controls, but similar between the diabetic groups. Diabetes in spontaneously hypertensive rats lowered renal tissue angiotensin-converting enzyme activity (40%), which was reduced further when higher ramipril doses were used. Diabetes associated with hypertension raised GLUT1 by 28% (P < 0.0001) and GLUT2 by 76% (P = 0.01), and both doses of ramipril equally reduced cortical GLUT1 (D vs D1 and vs D0.01, P ≤ 0.001). GLUT2 levels were reduced in D0.01 (P < 0.05 vs D). Diabetes increased urinary albumin and TGF-β1 urinary excretion, but the 15-day ramipril treatment (with either dose) did not reduce them. In conclusion, ramipril is effective in lowering renal tissue angiotensin-converting enzyme activity, as well as blocking cortical GLUT1 overexpression, which may be beneficial in arresting the development of diabetic nephropathy.