Balance of growth factors in the human perinatal lung : Implications for physiological lung development and link to bronchopulmonary dysplasia

The aims of this Thesis was to evaluate the role of proangiogenic placental growth factor (PlGF), antiangiogenic endostatin and lymphangiogenic vascular endothelial growth factor (VEGF) -C as well as the receptors vascular endothelial growth factor receptor (VEGFR) -2 and VEGFR-3 during lung development and in development of lung injury in preterm infants. The studied growth factors were selected due to a close relationship with VEGF-A; a proangiogenic growth factor important in normal lung angiogenesis and lung injury in preterm infants. The thesis study consists of three analyses. I: Lung samples from fetuses, preterm and term infants without lung injury, as well as preterm infants with acute and chronic lung injury were stained by immunohistochemistry for PlGF, endostatin, VEGF-C, VEGFR-2 and VEGFR-3. II: Tracheal aspirate fluid (TAF) was collected in the early postnatal period from a patient population consisting of 59 preterm infants, half developing bronchopulmonary dysplasia (BPD) and half without BPD. PlGF, endostatin and VEGF-C concentrations were measured by commercial enzyme-linked immunosorbent assay (ELISA). III: Cord plasma was collected from very low birth weight (VLBW) (n=92) and term (n=48) infants in conjuncture with birth and endostatin concentrations were measured by ELISA. I: All growth factors and receptors studied were consistently stained in immunohistochemistry throughout development. For endostatin in early respiratory distress syndrome (RDS), no alveolar epithelial or macrophage staining was seen, whereas in late RDS and BPD groups, both alveolar epithelium and macrophages stained positively in approximately half of the samples. VEGFR-2 staining was fairly consistent, except for the fact that capillary endothelial staining in the BPD group was significantly decreased. II: During the first postnatal week in TAF mean PlGF concentrations were stable whereas mean endostatin and VEGF-C concentrations decreased. Higher concentrations of endostatin and VEGF-C correlated with lower birth weight (BW) and associated with administration of antenatal betamethasone. Parameters reflecting prenatal lung inflammation associated with lower PlGF, endostatin and VEGF-C concentrations. A higher mean supplemental fraction of inspired oxygen during the first 2 postnatal weeks (FiO2) correlated with higher endostatin concentrations. III: Endostatin concentrations in term infants were significantly higher than in VLBW infants. In VLBW infants higher endostatin concentrations associated with the development of BPD, this association remained significant after logistic regression analysis. We conclude that PlGF, endostatin and VEGF-C all have a physiological role in the developing lung. Also, the VEGFR-2 expression profile seems to reflect the ongoing differentiation of endothelia during development. Both endostatin and VEGFR-2 seem to be important in the development of BPD. During the latter part of the first postnatal week, preterm infants developing BPD have lower concentrations of VEGF-A in TAF. Our findings of disrupted VEGFR-2 staining in capillary and septal endothelium seen in the BPD group, as well as the increase in endostatin concentrations both in TAF and cord plasma associated with BPD, seem to strengthen the notion that there is a shift in the angiogenic balance towards a more antiangiogenic environment in BPD. These findings support the vascular hypothesis of BPD.

Matrix proteinases in lung injury in the preterm infant

During inflammation, excess production and release of matrix proteinases, including matrix metalloproteinases (MMPs) and serine proteinases, may result in dysregulated extracellular proteolysis leading to development of tissue damage. Pulmonary inflammation may play an important role in the pathogenesis of lung injury in the preterm infant. The aims of this study were to evaluate involvement of MMPs and serine proteinase trypsin in acute and chronic lung injury in preterm infants and to study the role of these enzymes in acute lung injury by means of an animal model of hyperoxic lung injury. Molecular forms and levels of MMP-2, -8, and -9, and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, as well as trypsin were studied in tracheal aspirate fluid (TAF) samples collected from preterm infants with respiratory distress. Expression and distribution of trypsin-2 and proteinase-activated receptor 2 (PAR2) was examined in autopsy lung specimens from fetuses, from preterm infants with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), and from newborn infants without lung injury. We detected higher MMP-8 and trypsin-2 and lower TIMP-2 in TAF from preterm infants with more severe acute respiratory distress. Infants subsequently developing BPD had higher levels of MMP-8 and trypsin-2 early postnatally than did those who survived without this chronic lung injury. Immunohistochemically, trypsin-2 was mainly detectable in bronchial epithelium, but also in alveolar epithelium, and its expression was strongest in prolonged RDS. Since trypsin-2 is potent activator of PAR2, a G-protein coupled receptor involved in inflammation, we studied PAR2 expression in the lung. PAR2 co-localized with trypsin-2 in bronchoalveolar epithelium and its expression was significantly higher in bronchoalveolar epithelium in preterm infants with prolonged RDS than in newborn controls. In the experimental study, rats were exposed to >95% oxygen for 24, 48, and 60 hours, or room air. At 48 hours of hyperoxia, MMP-8 and trypsin levels sharply increased in bronchoalveolar lavage fluid, and expression of trypsin appeared in alveolar epithelium, and MMP-8 predominantly in macrophages. In conclusion, high pulmonary MMP-8 and trypsin-2 early postnatally are associated with severity of acute lung injury and subsequent development of BPD in preterm infants. In the injured preterm lung, trypsin-2 co-localizes with PAR2 in bronchoalveolar epithelium, suggesting that PAR2 activated by high levels of trypsin-2 is involved in lung inflammation associated with development of BPD. Marked increase in MMP-8 and trypsin early in the course of experimental hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury. Further exploration of the roles of trypsin and MMP-8 in lung injury may offer new targets for therapeutic intervention.

Porlyl oligopeptidase in development

Prolyl oligopeptidase (POP, prolyl endopeptidase, EC 3.4.21.26) is a serine-type peptidase (family S9 of clan SC) hydrolyzing peptides shorter than 30 amino acids. POP has been found in various mammalian and bacterial sources and it is widely distributed throughout different organisms. In human and rat, POP enzyme activity has been detected in most tissues, with the highest activity found mostly in the brain. POP has gained scientific interest as being involved in the hydrolyzis of many bioactive peptides connected with learning and memory functions, and also with neurodegenerative disorders. In drug or lesion induced amnesia models and in aged rodents, POP inhibitors have been able to revert memory loss. POP may have a fuction in IP3 signaling and it may be a possible target of mood stabilizing substances. POP may also have a role in protein trafficking, sorting and secretion. The role of POP during ontogeny has not yet been resolved. POP enzyme activity and expression have shown fluctuation during development. Specially high enzyme activities have been measured in the brain during early development. Reduced neuronal proliferation and differentation in presence of POP inhibitor have been reported. Nuclear POP has been observed in proliferating peripheral tissues and in cell cultures at the early stage of development. Also, POP coding mRNA is abundantly expressed during brain ontogeny and the highest levels of expression are associated with proliferative germinal matrices. This observation indicates a special role for POP in the regulation of neurogenesis during development. For the experimental part, the study was undertaken to investigate the expression and distribution of POP protein and enzymatic activity of POP in developing rat brain (from embryonic day 14 to post natal day 7) using immunohistochemistry, POP enzyme activity measurements and western blot-analysis. The aim was also to find in vivo confirmation of the nuclear colocalization of POP during early brain ontogeny. For immunohistochemistry, cryosections from the brains of the fetuses/rats were made and stained using specific antibody for POP and fluorescent markers for POP and nuclei. The enzyme activity assay was based on the fluorescence of 7- amino-4-methylcoumarin (AMC) generated from the fluorogenic substrate succinyl-glycyl-prolyl-7-amino-4-methylcoumarin (Suc-Gly-Pro-AMC) by POP. The amounts of POP protein and the specifity of POP antibody in rat embryos was confirmed by western blot analysis. We observed that enzymatic activity of POP is highest at embryonic day 18 while the protein amounts reach their peak at birth. POP was widely present throughout the developmental stages from embryonic day 14 to parturition day, although the POP-immunoreactivity varied abundantly. At embryonic days 14 and 18 notably amounts of POP was distributed at proliferative germinal zones. Furthermore, POP was located in the nucleus early in the development but is transferred to cytosol before birth. At P0 and P7 the POP-immunoreactivity was also widely observed, but the amount of POP was notably reduced at P7. POP was present in cytosol and in intercellular space, but no nuclear POP was observed. These findings support the idea of POP being involved in specific brain functions, such as neuronal proliferation and differentation. Our results in vivo confirm the previous cell culture results supporting the role of POP in neurogenesis. Moreover, an inconsistency of POP protein amounts and enzymatic activity late in the development suggests a strong regulation of POP activity and a possible non-hydrolytic role at that stage.

Human parvovirus infections during pregnancy : Special reference to the child-care employees

Human parvovirus B19 (B19V) is known to cause anemia, hydrops fetalis, and fetal death especially during the first half of pregnancy. Women who are in occupational contact with young children are at increased risk of B19V infection. The role of the recently discovered human parvovirus, human bocavirus (HBoV), in reproduction is unknown. The aim of this research project was to establish a scientific basis for assessing the work safety of pregnant women and for issuing special maternity leave regulations during B19V epidemics in Finland. The impact of HBoV infection on the pregnant woman and her fetus was also defined. B19V DNA was found in 0.8% of the miscarriages and in 2.4% of the intrauterine fetal death (IUFD; fetal death after completed 22 gestational weeks). All control fetuses (from induced abortions) were B19V-DNA negative. The findings on hydropic B19V DNA-positive IUFDs with evidence of acute or recent maternal B19V infection are in line with those of previous Swedish studies. However, the high prevalence of B19V-related nonhydropic IUFDs noted in the Swedish studies was mostly without evidence of maternal B19V infection and was not found during the third trimester. HBoV was not associated with miscarriages or IUFDs. Almost all of the studied pregnant women were HboV-IgG positive, and thus most probably immune to HBoV. All preterm births, perinatal deaths, smallness for gestational age (SGA) and congenital anomaly were recorded among the infants of child-care employees in a nationwide register-based cohort study over a period of 14 years. Little or no differences in the results were found between the infants of the child-care employees and those of the comparison group. The annual B19V seroconversion rate was over two-fold among the child-care employees, compared to the women in the comparison group. The seropositivity of the child-care employees increased with age, and years from qualification/joining the trade union. In general, the child-care employees are not at increased risk for adverse pregnancy outcome. However, at the population level, the risk of rare events, such as adverse pregnancy outcomes attributed to infections, could not be determined. According to previous studies, seronegative women had a 5 10% excess risk of losing the fetus during the first half of their pregnancy, but thereafter the risk was very low. Therefore, an over two-fold increased risk of B19V infection among child-care employees is considerable, and should be taken into account in the assessment of the occupational safety of pregnant women, especially during the first half of their pregnancy.

Towards Optimal Testing of Auditory Memory : Methodological development of recording of the mismatch negativity (MMN) of the auditory event-related potential (ERP)

The overlapping sound pressure waves that enter our brain via the ears and auditory nerves must be organized into a coherent percept. Modelling the regularities of the auditory environment and detecting unexpected changes in these regularities, even in the absence of attention, is a necessary prerequisite for orientating towards significant information as well as speech perception and communication, for instance. The processing of auditory information, in particular the detection of changes in the regularities of the auditory input, gives rise to neural activity in the brain that is seen as a mismatch negativity (MMN) response of the event-related potential (ERP) recorded by electroencephalography (EEG). --- As the recording of MMN requires neither a subject s behavioural response nor attention towards the sounds, it can be done even with subjects with problems in communicating or difficulties in performing a discrimination task, for example, from aphasic and comatose patients, newborns, and even fetuses. Thus with MMN one can follow the evolution of central auditory processing from the very early, often critical stages of development, and also in subjects who cannot be examined with the more traditional behavioural measures of auditory discrimination. Indeed, recent studies show that central auditory processing, as indicated by MMN, is affected in different clinical populations, such as schizophrenics, as well as during normal aging and abnormal childhood development. Moreover, the processing of auditory information can be selectively impaired for certain auditory attributes (e.g., sound duration, frequency) and can also depend on the context of the sound changes (e.g., speech or non-speech). Although its advantages over behavioral measures are undeniable, a major obstacle to the larger-scale routine use of the MMN method, especially in clinical settings, is the relatively long duration of its measurement. Typically, approximately 15 minutes of recording time is needed for measuring the MMN for a single auditory attribute. Recording a complete central auditory processing profile consisting of several auditory attributes would thus require from one hour to several hours. In this research, I have contributed to the development of new fast multi-attribute MMN recording paradigms in which several types and magnitudes of sound changes are presented in both speech and non-speech contexts in order to obtain a comprehensive profile of auditory sensory memory and discrimination accuracy in a short measurement time (altogether approximately 15 min for 5 auditory attributes). The speed of the paradigms makes them highly attractive for clinical research, their reliability brings fidelity to longitudinal studies, and the language context is especially suitable for studies on language impairments such as dyslexia and aphasia. In addition I have presented an even more ecological paradigm, and more importantly, an interesting result in view of the theory of MMN where the MMN responses are recorded entirely without a repetitive standard tone. All in all, these paradigms contribute to the development of the theory of auditory perception, and increase the feasibility of MMN recordings in both basic and clinical research. Moreover, they have already proven useful in studying for instance dyslexia, Asperger syndrome and schizophrenia.

Maturation of a transient outward potassium current in mouse fetal hypothalamic neurons in culture

The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypothalamic neurons developing in culture medium from days 1 to 17. The neurons were derived from fetuses of IOPS/OF1 mice on the 14th day of gestation. The mature neurons (>six days in culture) showed both a transient potassium current and a non-inactivating delayed rectifier potassium current. These were identified pharmacologically by using the potassium channel blockers tetraethyl ammonium chloride and 4-aminopyridine, and on the basis of their kinetics and voltage sensitivities. The delayed rectifier potassium current had a threshold of −20 mV, a slow time-course of activation, and was sustained during the voltage pulse. The 4-aminopyridine-sensitive current was transient, and was activated from a holding potential more negative (−80 mV) than that required for evoking the delayed rectifier potassium current (−40 mV). The delayed rectifier potassium current was detectable from day 1 onwards, while the transient potassium current showed a distinct developmental trend. The time-constant of inactivation became faster with age in culture. The half steady-state inactivation potential showed a shift towards less negative membrane potentials with age, and the relationship was best described by a logarithmic regression equation.The developmental trend of the transient potassium current may relate functionally to the progressive morphological changes, and the appearance of synaptic connections during ontogenesis.

Estudo estrutural do pênis de fetos humanos normais e anencéfalos no segundo trimestre de gestação

A anecencefalia é o Defeito do Tubo Neural (DTN) mais severo em fetos humanos. Há uma demanda crescente para reposição tissular em doenças crônicas e cirurgias reconstrutoras. Tecidos fetais têm sido utilizados como substitutos para órgãos sólidos. Comparar a estrutura e morfologia do corpo cavernoso e corpo esponjoso de pênis de fetos humanos anencéfalos e de controle a fim de propor um novo modelo para estudos biológicos e transplantes teciduais. Foram estudados 11 pênis de fetos de controle de 14 a 23 Semanas Pós Concepção (SPC), e cinco pênis de fetos anencéfalos de 18 a 22 SPC. Os órgãos foram removidos e processados pelas técnicas histo e imunohistoquímicas rotineiras. A análise do tecido conjuntivo, células musculares lisas e fibras elásticas foram realizadas em lâminas dos espécimes. Os dados foram expressos em Densidade de àrea (Da) utilizando-se um software de processamento digital. As médias foram comparadas utilizando-se o Teste - T não pareado e quando aplicável, a regressão linear simples foi utilizada. Foi considerada significância estatística se p<0,05. O septo intercavernoso encontrava-se presente em todas as amostras. Não foram observadas diferenças da Da do tecido colágeno e musculatura lisa dos pênis de fetos anencéfalos quando comparados aos normais. A regressão linear simples sugere que durante o desenvolvimento humano há um aumen2to gradual do tecido colágeno (R2=0,45) e uma diminuição da musculatura lisa (R =0,62) no corpo cavernoso de ambos os grupos. A elastina encontrava-se presente apenas em fetos a partir da 20 SPC. Não houve diferença na estrutura da genitália entre fetos normais e enencéfalos. Apresença da elastina em fetos a partir da 20 SPC é um dado objetivo da manutenção da capacidade de ereção nestes grupos. A histo e imunohistoquímica sugerem que o desenvolvimento do pênis destes fetos encontra-se inalterado. Futuros estudos deverão ser realizados com o objetivo de avaliar fetos anencéfalos como um potencial grupo de doadores teciduais e um adequado modelo para estudos biológicos.

Defeitos do tubo neural causam alterações estruturais no ureter? Estudo em fetos humanos

Anencefalia é o defeito do tubo neural mais severo. A morfologia do ureter de fetos anencéfalos é desconhecida. O objetivo deste trabalho é analisar a estrutura do ureter de fetos humanos normais e anencéfalos (FHA). Nós estudamos 16 ureteres de 8 fetos sem anomalias congênitas (4 masculinos e 4 femininos) com idades entre 16 e 27 semanas pós concepção (SPC) e 14 ureteres de 7 FHA (4 masculinos e 3 femininos) com idades entre 19 e 33 SPC. Os ureteres foram dissecados e emblocados em parafina. Foram feitos cortes com 5 m e depois corados com Tricrômico de Masson, para quantificação das células de músculo liso (CML) e determinação da área da a luz do ureter, espessura e diâmetro. As amostras também foram coradas com Resorcina Fucsina de Weigert ( para observação das fibras elásticas) e Vermelho de Picro Sirius com polarização e análise imunohistoquímica das fibras do colágeno tipo III. Os dados da quantificação do músculo foram expressos em densidade volumétrica (Vv-%). As imagens foram capturadas com microscópio Olympus BX51 e câmera Olympus DP70. A análise morfológica da área do lúmen, espessura e diâmetro foram feitas usando o software Image J. As médias foram comparadas usando o teste t não pareado (p<0.05). O epitélio do ureter estava bem preservado em ambos os grupos, e não houve diferença entre os grupos. Não observamos fibras do sistema elástico em qualquer ureter analisados. Concentração de músculo liso (Vv) não diferiram significativamente (p = 0,4413) em FHA (12% 1,628) e grupo controle (13,51% 0,9231). A área de luz ureteral foi significativamente menor (p = 0,0341) em FHA (6365μm 1,282), quando comparado ao grupo controle (20,170 5,480 mM). O diâmetro ureteral foi significativamente menor (p = 0,0294) em FHA (166.7μm 10,99) quando comparado ao grupo controle (240 26,6 mM). A espessura ureteral foi significativamente menor (p = 0,0448) em FHA (30.57μm 2,034), quando comparado ao grupo controle (7,453 47.49μm). Colágeno tipo III foi observado em maior quantidade nos ureteres da FHA. Alterações estruturais ureterais nos fetos anencéfalos foram significativas em nosso estudo. O ureter de fetos com anencefalia mostraram mais concentração de colágeno tipo III, menor diâmetro, área e espessura. Nervos ureterais em FHA podem ser modificados devido a lesão cerebral com consequente dano no controle dos nervos ureterais. Isto pode levar a alterações estruturais no ureter de fetos anencéfalos.

Desenvolvimento do pênis durante o período fetal humano (13 a 36 semanas pós-concepção)

Objetivo: Analisar, em fetos humanos, o crescimento da área do pênis, da túnica albugínea e das estruturas eréteis (corpos cavernosos e corpo esponjoso), bem como o aparecimento e modificações das principais estruturas que compõem estes tecidos (colágeno, músculo liso e fibra elástica) durante o período fetal (13 a 36 semanas pós-concepção), fornecendo padrões normativos de crescimento. Material e Métodos: Foram utilizados 56 fetos humanos do sexo masculino com idade gestacional compreendida entre 13 e 36 semanas pós-concepção (SPC). Foram utilizadas técnicas histoquímicas, imunohistoquímicas, e análises morfométricas, e analisados os seguintes parâmetros: área total do pênis, área do corpo cavernoso, área do corpo esponjoso e a espessura da túnica albugínea na região dorsal e ventral do corpo cavernoso. No corpo cavernoso e no corpo esponjoso, as fibras musculares, o colágeno e as fibras do sistema elástico, foram identificados e quantificados por percentagem, no programa Image J (NIH, Bethesda, EUA).Resultados: Da 13 à 36 semana pós-concepção, a área do pênis variou de 0,95mm2 a 24,25mm2. No mesmo período a área do corpo cavernoso variou de 0,28mm2 a 9,12mm2 e a área do corpo esponjoso de 0,14mm2 a 3,99mm2. No corpo cavernoso a percentagem de fibras colágenas, fibras musculares e fibras do sistema elástico variaram, respectivamente, de 19,88% a 36,60%, de 4,39 % a 29,76 % e de 1,91% a 8,92%. No corpo esponjoso a percentagem de fibras colágenas, fibras musculares e fibras do sistema elástico variaram, respectivamente de 34,65% a 45,89%, de 0,60% a 11,90% e de 3,22% a 11,93%. A espessura da túnica albugínea variou de 0,029 a 0,296 na região dorsal e de 0,014 a 0,113 na região ventral do corpo cavernoso.Conclusão: Existe correlação fortemente positiva entre o crescimento da área total, da área do corpo cavernoso e da área do corpo esponjoso, com a idade gestacional, assim como existe correlação entre o crescimento dos elementos constituintes do tecido erétil do pênis (colágeno, fibras musculares lisas e fibras elásticas) com a idade gestacional no período fetal estudado. O ritmo de crescimento do pênis é mais intenso no IIo. trimestre gestacional (13 a 24 SPC). O crescimento da espessura da túnica albugínea também foi diretamente proporcional e apresentou correlação fortemente positiva com a idade gestacional, sendo maior na região dorsal em relação à região ventral do corpo cavernoso.

Papel do sistema renina-angiotensina e do desequilíbrio redox em modelo experimental de pré-eclâmpsia induzida por L-NAME

Pré-eclâmpsia (PE), uma síndrome sistêmica da gestação caracterizada por proteinúria e hipertensão, está associada a uma significativa mortalidade tanto materna quanto fetal. Eentretanto, sua fisiopatologia ainda não é totalmente compreendida. Apesar de um expressivo aumento da atividade do sistema renina-angiotensina (SRA) na gestação normal, a pressão arterial não aumenta. Além disso, a redução da pressão de perfusão intra-uterina promove um aumento na liberação de espécies reativas de oxigênio que podem contribuir para a hipertensão na gestação. Dessa forma, o objetivo deste trabalho foi estudar o papel do SRA vascular, assim como, do estresse oxidativo plasmático, cardiorenal e placentário para a regulação cardiovascular materna em ratas normotensas e em modelo de PE induzida por L-NAME. Foi observado um aumento da pessão arterial em animais que receberal L-NAME. As ratas grávidas + L-NAME apresentaram um menor ganho de massa corporal durante a gestação, menor múmero de filhotes vivos, maior número de abortos, menor massa placentária total e fetos com menor massa corporal. Foi observada uma redução na resposta vasodilatadora induzida por acetilcolina (ACh) e angiotensina (Ang) II, aumento na resposta vasodilatadora induzida por nitroglicerina (NG) e aumento na resposta vasoconstritora induzida por fenilefrina (Phe) e Ang II em LAM de ratas grávida e não grávidas que receberam L-NAME. Não foi observada diferença na expressão dos receptores AT1 e AT2 e das enzimas ECA, ECA2 e eNOS. Foi observado um aumento na concentração plasmática de renina e bradicinina (BK) em ratas grávidas + L-NAME e uma redução na concentração de Ang 1-7. As ratas grávidas e grávidas + L-NAME apresentaram um aumento nos níveis séricos de estradiol. Os níveis de malondialdeído e carbonilação de proteínas estava aumentados e a atividade das enzimas antioxidantes SOD e GPx estavam reduzidas em ratas grávidas e não grávidas que receberam L-NAME. A atividade da CAT não apresentou diferença entre os grupos. Em conclusão, uma redução na vasodilatação induzida pela Ang II associada a um aumento da vasoconstrição promovida por este peptídeo, sugerem uma contribuição do SRA no desenvolvimento das complicações características da PE observadas no modelo experimental de PE induzido por L-NAME. Do mesmo modo, a peroxidação lipídica e oxidação de proteínas aumentadas, assim como, as atividades enzimáticas antioxidantes reduzidas sugerem a contribuição de uma defesa antioxidante comprometida e um dano oxidativo aumentado para o desenvolvimento da hipertensão e disfunção endotelial, aumento da mortalidade fetal e retardo do crescimento intra-uterino observados no modelo de PE estudado.

Efeitos do controle de temperatura e da vimpocetina sobre os déficits neurocomportamentais decorrentes do estresse por separação materna em camundongos suíços neonatos

O estresse durante o desenvolvimento está associado com diversas desordens neurocomportamentais, que podem persistir ao longo da vida. A hiperatividade é um dos transtornos comportamentais que, com maior frequência, observa-se em humanos submetidos ao estresse precoce. Esse transtorno pode ser a manifestação clínica predominante, ou mesclar-se com déficit de atenção, impulsividade e retardo da aprendizagem, constituindo o Transtorno de Déficit de Atenção e Hiperatividade (TDAH), com número de casos diagnosticados em ascensão. Diversos protocolos experimentais utilizam a separação materna (SM) de roedores neonatos para mimetizar as consequências do estresse precoce em humanos. Esta predileção por roedores recém-nascidos se deve à sua equivalência aproximada com fetos humanos no terceiro trimestre da gestação em termos de neurodesenvolvimento, quando ocorre o maior crescimento do Sistema Nervoso Central fetal. Neste trabalho, camundongos suíços neonatos foram submetidos a sessões diárias de isolamento com separação materna, entre o 2 e o 10 dias de vida pós-natal (PN2 a PN10), variando-se a temperatura de isolamento dos filhotes, que permaneciam sem aquecimento (na temperatura do biotério, entre 22 e 25C) ou eram mantidos aquecidos a 37C durante essas sessões. Portanto, foram três grupos experimentais: isolamento aquecido com SM; isolamento não aquecido com SM; e controle. Os animais do grupo controle foram pesados em PN2 e PN10 e, prontamente, devolvidos às progenitoras. Todos os animais foram desmamados e sexados em PN21, não sendo perturbados até a realização dos testes neurocomportamentais, a partir de PN30, que incluíram os Testes de Campo Aberto e de Esquiva Inibitória. Num segundo estudo, foram realizadas dosagens séricas da corticosterona basal e dos hormônios tireoidianos nos três grupos experimentais, em PN6, PN10 e PN30. Finalmente, num terceiro estudo, camundongos do grupo controle e do grupo submetido ao isolamento não aquecido com SM foram tratados com vimpocetina (20g/kg), um neuroprotetor potencial, ou com veículo (Dimetilsulfóxido), a fim de avaliar os efeitos da vimpocetina na atividade locomotora. O 1 estudo demonstrou que a temperatura foi um fator crítico para a manifestação de hiperatividade locomotora no Teste do Campo Aberto, que ocorreu, somente, nos animais do grupo submetido ao isolamento não aquecido. Adicionalmente, não houve diferenças entre os grupos experimentais no Teste da Esquiva Inibitória, quanto à memória e à aprendizagem. O 2 estudo demonstrou que a temperatura do isolamento influenciou os níveis da corticosterona basal e dos hormônios tireoidianos em PN10 e em PN30. No 3 estudo, a vimpocetina reduziu a hiperatividade locomotora no grupo de animais submetidos ao isolamento não aquecido com SM. De todo o exposto, conclui-se que o isolamento com SM de camundongos suíços neonatos à temperatura de 22 a 25C aumentou a atividade locomotora nesses animais, e que a vimpocetina foi capaz de atenuar esse comportamento, sem aumento da mortalidade. Finalmente, considerando-se o maior risco de desenvolvimento do TDAH em crianças e adolescentes com históricos de prematuridade, levanta-se a hipótese de que o estresse térmico pelo frio seja um dos fatores envolvidos na fisiopatogenia da hiperatividade nesses casos.

Avaliação dos testículos de ratos submetidos à torção testicular antes, durante e após a puberdade, e o efeito do tratamento com L-arginina

Torção testicular (TT) é uma síndrome urológica comumente encontrada em recém nascidos, crianças e adolescentes. Neste trabalho foi estudada as lesões morfológicas e a função reprodutiva em ratos adultos que sofreram TT, em diferentes idades de maturidade sexual e o efeito protetor da L-arginina, contra os danos causados pela isquemia/reperfusão na torção testicular. Dezoito ratos pré-púberes (4 semanas de vida) dezessete púberes (6 semanas de vida) e dezessete adultos (9 semanas de vida) foram submetidos à TT. Sob anestesia o testículo direito foi rotacionado em 720 e fixado, sendo então destorcido após 4 horas. Vinte e quatro ratos (ARG4, n=8, ARG6, n=8 e ARG9 n=8) foram submetidos ao tratamento com 650mg/kg de L-arginina, por via oral, durante 7 dias. Outros trinta ratos de mesma idade sofreram cirurgia simulada (SH4, n=10, SH6, n=10 e SH9, n=10). Com 12 semanas de idade, foram submetidos ao acasalamento controlado com 3 fêmeas e ao vigésimo dia de gestação o número de fetos, corpos lúteos, absorções e implatações foram contados. Na 14 semana, os ratos foram mortos e os espermatozóides coletados da cauda dos epidídimos, foi anotado o peso corporal, o peso e volume testicular. O soro foi usado para dosagem de testosterona. Foram avaliadas a concentração, motilidade e a viabilidade espermática. Os testículos coletados foram fixados em Bouin, pós-fixados em formalina e processados em parafina. Utilizando o programa de imagem Image J, lâminas coradas com HE, mensuramos a altura do epitélio, densidade volumétrica e diâmetro do túbulo seminífero. Para avaliar a integridade do epitélio seminífero foi utilizado a frequência dos estágios do ciclo do epitélio e o escorre de Johnsen. Também foi avaliado a proliferação do compartimento tubular e intertubular, através da imunomarcação com PCNA. Os dados foram tabulados e as médias dos grupos comparadas pelo teste de ANOVA com pós-teste de Bonferroni ou teste de Kruskal-Wallis com pós teste de Dunns (programa Graphpad Prism, com p < 0,05). Os resultados revelaram grandes danos produzidos pela injuria testicular, no testículo ipsilateral, com diminuição da capacidade reprodutiva, da concentração, viabilidade e mobilidade, do peso e volume testicular. Animais TT9 não apresentaram espermatozoides nas amostras coletas. Houve diminuição do diâmetro dos túbulos seminíferos e da altura do epitélio, aumento do compartimento intertubular, com ênfase dos vasos sanguíneos, aumento da proliferação celular estromal e diminuição da proliferação epitelial. Houve diminuição da concentração sérica de testosterona no grupo TT4, quando comparado como grupo TT9. Em geral, os animais submetidos à torção na fase adulta foram os mais acometidos. Não foram encontradas alterações dignas de nota, nos testículo contralaterais. Animais tratados com L-arginina obtiveram melhora dos índices reprodutivos, com aumento da potência em todas as idades. Houve aumento da concentração espermática no testículo contralateral de ARG4 e ARG6, mostrando que a L-arginina atuou como antioxidante. Não houve proteção para as lesões causadas pela torção na maioria dos grupos, mas os animais tratados ARG9 apresentaram concentração espermática mensuravel, quando comparados aos ratos TT9, que tinham azospermia.

Self-organization of reflexive behavior from spontaneous motor activity.

In mammals, the development of reflexes is often regarded as an innate process. However, recent findings show that fetuses are endowed with favorable conditions for ontogenetic development. In this article, we hypothesize that the circuitry of at least some mammalian reflexes can be self-organized from the sensory and motor interactions brought forth in a musculoskeletal system. We focus mainly on three reflexes: the myotatic reflex, the reciprocal inhibition reflex, and the reverse myotatic reflex. To test our hypothesis, we conducted a set of experiments on a simulated musculoskeletal system using pairs of agonist and antagonist muscles. The reflex connectivity is obtained by producing spontaneous motor activity in each muscle and by correlating the resulting sensor and motor signals. Our results show that, under biologically plausible conditions, the reflex circuitry thus obtained is consistent with that identified in relation to the analogous mammalian reflexes. In addition, they show that the reflex connectivity obtained depends on the morphology of the musculoskeletal system as well as on the environment that it is embedded in.

Twitching in sensorimotor development from sleeping rats to robots.

It is still not known how the 'rudimentary' movements of fetuses and infants are transformed into the coordinated, flexible and adaptive movements of adults. In addressing this important issue, we consider a behavior that has been perennially viewed as a functionless by-product of a dreaming brain: the jerky limb movements called myoclonic twitches. Recent work has identified the neural mechanisms that produce twitching as well as those that convey sensory feedback from twitching limbs to the spinal cord and brain. In turn, these mechanistic insights have helped inspire new ideas about the functional roles that twitching might play in the self-organization of spinal and supraspinal sensorimotor circuits. Striking support for these ideas is coming from the field of developmental robotics: when twitches are mimicked in robot models of the musculoskeletal system, the basic neural circuitry undergoes self-organization. Mutually inspired biological and synthetic approaches promise not only to produce better robots, but also to solve fundamental problems concerning the developmental origins of sensorimotor maps in the spinal cord and brain.

Pancreatic and adrenal development and function in an ovine model of polycystic ovary syndrome.

Polycystic Ovary Syndrome (PCOS) is a complex disorder encompassing reproductive and metabolic dysfunction. Ovarian hyperandrogenism is an endocrine hallmark of human PCOS. In animal models, PCOS-like abnormalities can be recreated by in utero over-exposure to androgenic steroid hormones. This thesis investigated pancreatic and adrenal development and function in a unique model of PCOS. Fetal sheep were directly exposed (day 62 and day 82 of gestation) to steroidal excesses - androgen excess (testosterone propionate - TP), estrogen excess (diethylstilbestrol - DES) or glucocorticoid excess (dexamethasone - DEX). At d90 gestation there was elevated expression of genes involved in β- cell development and function: PDX-1 (P<0.001), and INS (P<0.05), INSR (P<0.05) driven by androgenic excess only in the female fetal pancreas. β- cell numbers (P<0.001) and in vitro insulin secretion (P<0.05) were also elevated in androgen exposed female fetuses. There was a significant increase in insulin secreting β-cell numbers (P<0.001) and in vivo insulin secretion (glucose stimulated) (P<0.01) in adult female offspring, specifically associated with prenatal androgen excess. At d90 gestation, female fetal adrenal gene expression was perturbed by fetal estrogenic exposure. Male fetal adrenal gene expression was altered more dramatically by fetal glucocorticoid exposure. In female adult offspring from androgen exposed pregnancies there was increased adrenal steroidogenic gene expression and in vivo testosterone secretion (P<0.01). This highlights that the adrenal glands may contribute towards excess androgen secretion in PCOS, but such effects might be secondary to other metabolic alterations driven by prenatal androgen exposure, such as excess insulin secretion Thus there may be dialogue between the pancreas and adrenal gland, programmed during early life, with implications for adult health Given both hyperinsulinaemia and hyperandrogenism are common features in PCOS, we suggest that their origins may be at least partially due to altered fetal steroidal environments, specifically excess androgenic stimulation