Resumo não disponível. / Malnutrition is a worldwide problem affecting millions of unborn and young children during the most vulnerable stages of brain development (1). All restriction of protein during the perinatal period of life can alter the development of mammalian fetus and have marked repercussions on development of the Central Nervous System (CNS). The brain is vulnerable to protein malnutrition with altered morphologic and biochemical maturation, leading to impaired functions. The focus of this study is to investigate [U-14C]glycine metabolism in malnourished rats submitted to pre- and postnatal protein deprivation (diet: 8% protein with addition and without addition of L-methionine) on glycine metabolism of rats (normonourished group: 25% protein). It was observed that protein malnutrition alters oxidation to CO2, conversion to lipids and protein synthesis from [U-14C]glycine in cerebellum of malnourished rats without addition of L-methionine on a diet at 7 and 21 days of postnatal life. Our results also indicate that protein malnutrition causes a retardation in the normally ordered progression of brain development, and the malnourished groups have smaller cells, reduction in cell numbers and smaller cerebellar weight comparing to the control group.

Les mécanismes de neuroplasticité sont essentiels pour la mise en place et le renforcement des circuits neuronaux lors de périodes critiques du développement, et permettent au cerveau de s'adapter au cours des différentes étapes de la vie. Ces mécanismes varient avec l'âge, sont généralement plus difficile à activer chez l'adulte, et diminuent dans le cerveau âgé. La stimulation magnétique transcrânienne répétée (rTMS) est actuellement utilisée pour moduler l'excitabilité corticale et est décrite comme prometteuse dans le traitement de certains troubles neurologiques. La rTMS de faible intensité (LI-rTMS), ne déclenchant pas directement de potentiels d'action dans les neurones stimulés, a aussi montré des effets thérapeutiques, il est donc important de comprendre les effets biologiques de ces champs magnétiques d'intensités similaires à celles présentes dans les régions adjacentes à la région ciblée par la rTMS de haute intensité. Nous avons utilisé une stimulation magnétique focale de faible intensité (10 mT), ciblant le cervelet ainsi que la voie olivocérébelleuse chez la souris, afin d'aborder certaines de ces questions. Le cervelet est un modèle pertinent, en effet son développement, sa structure, son vieillissement et ses fonctions sont bien décrits, facilitant la détection d'éventuelles modifications dans cette région. Nous avons étudié les effets de LI-rTMS, in vivo ou in vitro, sur la morphologie neuronale, le comportement, et la plasticité post-lésionnelle. Dans une première étude nous avons montré que la LI-rTMS in vivo modifie les épines et la morphologie dendritique des cellules de Purkinje, ces modifications sont associées à une amélioration de la mémoire. / Neuroplasticity is essential for the establishment and strengthening of neural circuits during the critical period of development, and are required for the brain to adapt to its environment. The mechanisms of plasticity vary throughout life, are generally more difficult to induce in the adult brain, and decrease with advancing age. Repetitive transcranial magnetic stimulation (rTMS) is commonly used to modulate cortical excitability and shows promise in the treatment of some neurological disorders. Low intensity magnetic stimulation (LI-rTMS), which does not directly elicit action potentials in the stimulated neurons, have also shown some therapeutic effects, and it is important to determine the biological mechanisms underlying the effects of these low intensity magnetic fields, such as would occur in the regions surrounding the central high-intensity focus of rTMS. We have used a focal low-intensity magnetic stimulation (10mT) to address some of these issues in the mouse cerebellum and olivocerebellar path. The cerebellum model is particularly useful as its development, structure, ageing and function are well described which allows us to easily detect eventual modifications. We assessed effects of in vivo or in vitro LI-rTMS on neuronal morphology, behavior, and post-lesion plasticity. We first showed that LI-rTMS treatment in vivo alters dendritic spines and dendritic morphology, in association with improved spatial memory. These effects were age dependent. To optimize stimulation parameters in order to induce post-lesion reinnervation we used our in vitro model of post-lesion repair to systematically investigate the effects of different LI-rTMS stimulation patterns and frequencies. We showed that the pattern of stimulation is critical for allowing repair, rather than the total number of stimulation pulses. Finally, we looked for potential underlying mechanisms participating in the effects of the LI-rTMS, using mouse mutants in vivo or in vitro. We found that the cryptochromes, which have magnetoreceptor properties, must be present for the response to magnetic stimulation to be transduced into biological effects. The ensemble of our results indicate that the effects of LI-rTMS depend upon the presence of magnetoreceptors, the stimulation protocol, and the age of the animal suggesting that future therapeutic strategies must be adapted to the neuronal context in each individual person.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Hipóxia-isquemia (HI) perinatal é um problema de saúde pública, e ocorrem aproximadamente 1,5 casos de encefalopatias por HI por 1000 nascidos vivos. Dos que sobrevivem 25-60% sofrem de deficiências permanentes do desenvolvimento neurológico, incluindo paralisia cerebral, convulsões, retardo mental, e dificuldade de aprender. Neurônios e oligodendrócitos, especialmente os progenitores, são os mais afetados pela HI. Existem vários modelos de HI, no entanto, poucos levam em consideração as intercorrências maternas, a importância da atividade placentária, e as trocas entre mãe-filho, que são clinicamente observadas em humanos. Robinson estabeleceu um modelo de HI sistêmica pré-natal transitório, onde o fluxo das artérias uterinas da rata grávida era obstruído por 45 minutos no décimo oitavo dia (E18) de gestação. Neste modelo foram observadas alterações que são similares às observadas em cérebros humanos que passaram por hipóxia perinatal, dentre as quais foram relatados aumento no nível de apoptose. Caspase-3 é descrita como uma enzima que atua na apoptose, e é amplamente utilizada como marcador para células apoptóticas. Vários autores vêm mostrando, entretanto, que a enzima caspase-3 pode estar ativada para fins não apoptóticos. No modelo de HI sistêmica pré-natal, foram observados astrogliose na substância branca, morte de oligodendrócitos, lesão em axônios tanto na substância branca como no córtex cerebral, e danos motores. Pouco se sabe da influencia do insulto HI no desenvolvimento do cerebelo, considerando que o cerebelo junto com o córtex motor, contribui para o controle motor. O objetivo desse trabalho foi avaliar a distribuição da caspase-3 clivada durante o desenvolvimento do cerebelo em um modelo de HI pré-natal. Os resultados deste trabalho demonstram que as células caspase-3 clivadas apresentaram duas morfologias distintas em ambos os grupos. Uma onde a caspase-3 foi observada apenas no núcleo, oscilando entre células com imunorreatividade fraca a intensa, e de células com a presença da caspase-3 no corpo celular, nos prolongamentos condensados e presença de fragmentos ao redor do soma, morfologia típica de célula em apoptose. A HI pré-natal, assim como nos hemisférios cerebrais, levou ao aumento de células caspase-3 clivadas com morfologia de progenitores de oligodendrócitos no cerebelo do grupo HI em P2, mas não em P9 e P23. Também foi demonstrado que a HI pré-natal não levou a uma ativação da apoptose em oligodendrócitos, neurônios e microglia (identificados por seus respectivos marcadores, CNPase, NeuN e ED1) apresentando marcação no núcleos de células GFAP+, na substância branca, camada granular e nas células da glia de Bergmann, em P9 e P23 no cerebelo. Podemos concluir que a HI pré-natal aumentou o número de células imunorreativas para a caspase-3 em um período crítico do desenvolvimento da oligodendroglia no cerebelo, e que a diminuição de progenitores de oligodendrócitos no cerebelo decorrente do insulto pré-natal visto em trabalhos anteriores, pode estar relacionada a morte celular por apoptose, embora não se possa descartar a hipótese da participação dessas células que apresentam caspase-3 clivada em outros eventos não apoptóticos desencadeados pela hipóxia-isquemia. / Perinatal hypoxia ischemia (HI) is a public health problem: about 1.5 cases of encephalopathy occurred per 1000 newborn alive. Those who survive 25-60% suffered permanent deficiencies in neurological development, including cerebral palsy seizures, mental retardation and learning difficulties. Neurons and oligodendrocytes, especially the progenitors were the more affected in an HI insult. There are many HI models; however, few of them taken in account the maternal status, the placental activity and the transport of substances between mother and fetus that are clinically observed in humans. Robinson described a model of transitory prenatal systemic HI, in that uterine arteries were clamped for 45 minutes in the eighteenth gestational day (E18). In this model many impairments very similar to those observed in human brain that suffered HI were showed, between them the increase in apoptosis levels. Caspase-3 is related to apoptosis and is largely used as a marker for apoptotic cells. However, many authors have shown that caspase-3 activation may be related not only with apoptosis. In the prenatal systemic HI model were observed white matter astrogliosis, oligodendroglial death, axonal lesion both in white matter as in cerebral cortex as also motor deficits. Little is known about the effects of HI insult in developing cerebellum, which is a region that, together with motor cortex, participates in motor control. The aim of this work was to evaluate cleaved caspase-3 distribution during cerebellar development in a model of prenatal HI. Our results showed that cleaved caspase-3 positive cells showed two different pattern of distribution in both groups. One of them where the caspase-3 was observed only in the nucleus, presenting both low or intense immunoreactivity, and another one presenting the caspase-3 distributed in the cell bodies and in the condensed processes as also the presence of fragments around the soma, a typical morphology of apoptotic cell. Prenatal HI, as also in the cerebral hemispheres leads to an increase in the number of cleaved caspase-3 positive cells that showed oligodendroglial progenitor cell phenotype in the P2 cerebellum, but not in P9 or P23, in HI group. It was also showed that prenatal HI do not leads to Caspase-3 distribution in oligodendrocytes, neurons or microglia (identified by their respective markers, CNPase, NeuN and ED1) however is present in the nucleus of GFAP+ cells, in the white matter, granular layer and in Bergmann Glia at P9 an P23. We may conclude that prenatal HI increased the number of caspase-3 imunorreactive cells in a critical period for oligodendroglial differentiation in developing cerebellum, and that the oligodendroglial progenitors decrease previously showed in the cerebellum as consequence of HI insult may be related to apoptotic cell death, although we may not discard the hypothesis of caspase-3 immunoreactive cells may be related with non apoptotic events triggered by hypoxia-ischemia.

Made available in DSpace on 2016-01-13T13:27:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-05-22. Added 1 bitstream(s) on 2016-01-13T13:33:18Z : No. of bitstreams: 1 000855558.pdf: 2496353 bytes, checksum: afab1cef7d337a1187a0d63fa17d398d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: O cerebelo é suscetível aos efeitos lesivos do etanol em exposições agudas ou crônicas, em diferentes idades, podendo ocasionar alterações motoras, de aprendizado, cognitivas e favorecer a morte celular apoptótica. Nosso objetivo foi avaliar a apoptose nos neurônios de Golgi, Purkinje e células granulares do cerebelo de ratos adultos da variedade UChA. Metodologia: Os ratos foram divididos em dois grupos experimentais (20 ratos por grupo): um grupo controle, composto de animais UChA abstinentes e o grupo experimental, formado por ratos UChA, consumidores voluntários de etanol a 10% (˂ 2g etanol/Kg peso corpóreo/dia). Dos 65 dias aos 120 dias de idade, os animais receberam solução de etanol a 10%. Aos 120 dias, os animais foram anestesiados e preparados para eutanásia. O cerebelo foi então analisado por microscopia eletrônica de transmissão (MET), imunoistoquímica para apoptose, caspase-3 (próapoptótica), XIAP (antiapoptótica) e IGF-1R nos neurônios de Golgi, Purkinje e granulares. Avaliou-se também por RT-PCR os genes reguladores da caspase-3, XIAP e IGF-1R no córtex cerebelar dos grupos estudados. Resultados: Os animais UChA mostraram acúmulo de gotas lipídicas, presença de núcleos eletrodensos nos neurônios granulares e de Purkinje, desorganização do corpo medular do cerebelo com alterações na bainha de mielina no córtex cerebelar. Identificou-se também aumento significativo na imunomarcação de caspase-3 e XIAP nos neurônios de Purkinje e granulares. A fragmentação do DNA foi significativa nas células de Purkinje. O IGF-1R não apresentou alterações. Não houve diferenças significativas na expressão dos genes de caspase-3, XIAP, e o IGF-1R. Conclusão: O etanol induz apoptose nas células de Purkinje e granulares do cerebelo de ratos adultos UChA / Introduction: The cerebellum is susceptible to the harmful effects of ethanol in acute or chronic exposures at different ages, which may cause motor, learning and cognitive disorders and promote apoptotic cell death (PCD) as well. Our objective was to evaluate apoptosis in Golgi and Purkinje neurons and in granular cell disease of the cerebellum of adult rats of the type UChA. Methodology: The rats were divided in two experimental groups (20 rats per group): a control group, consisting of abstinent UChA animals and the experimental group, formed by UChA rats, volunteers consumers of 10% ethanol (˂ 2g ethanol / kg weight body / day). From 65 day-age to the 120 day-age, the animals received 10% ethanol solution. After 120 days, the rats were anesthetized and prepared for euthanasia. The cerebellum was then analyzed by transmission electron microscopy (TEM), immunohistochemistry to apoptosis, caspase-3 (pro-apoptotic), XIAP (anti-apoptotic) and IGF-1R in Golgi, Purkinje neurons and granular as well. It was also evaluated by RT-PCR (Reverse transcription polymerase chain reaction) the regulatory genes caspase-3, XIAP and IGF-1R in the cerebellar cortex of groups. Results: The UChA animals showed accumulation of lipid droplets, presence of electron-nuclei in granule neurons and Purkinje, disorganization of the medullary body of the cerebellum with some changes in the myelin sheath of the cortex. It was also identified significant increase in the Immunostaining caspase-3 and XIAP in Purkinje neurons and granular. The DNA fragmentation was significant in Purkinje cells. The IGF-1R didn't have any change though. There were no significant differences in the formulation of caspase-3 genes, XIAP and IGF-1R. Conclusion: Ethanol induces apoptosis in granular and Purkinje cells of the cerebellum of adult UChA rats / FAPESP: 2011/50466-0

Resumo não disponível. / Malnutrition is a worldwide problem affecting millions of unborn and young children during the most vulnerable stages of brain development (1). All restriction of protein during the perinatal period of life can alter the development of mammalian fetus and have marked repercussions on development of the Central Nervous System (CNS). The brain is vulnerable to protein malnutrition with altered morphologic and biochemical maturation, leading to impaired functions. The focus of this study is to investigate [U-14C]glycine metabolism in malnourished rats submitted to pre- and postnatal protein deprivation (diet: 8% protein with addition and without addition of L-methionine) on glycine metabolism of rats (normonourished group: 25% protein). It was observed that protein malnutrition alters oxidation to CO2, conversion to lipids and protein synthesis from [U-14C]glycine in cerebellum of malnourished rats without addition of L-methionine on a diet at 7 and 21 days of postnatal life. Our results also indicate that protein malnutrition causes a retardation in the normally ordered progression of brain development, and the malnourished groups have smaller cells, reduction in cell numbers and smaller cerebellar weight comparing to the control group.

Introdução: Há pouca informação a respeito do crescimento cerebelar em recém-nascidos de muito baixo peso (RNMBP). Tradicionalmente as funções cerebelares são relatadas como controle do tônus, postura e coordenação da atividade motora. Entretanto, há um incremento de evidências da implicação do cerebelo na cognição, linguagem, memória e aprendizagem motora. No pré-termo com seqüelas neurológicas, o cerebelo está relacionado com alterações cognitivas e distúrbios emocionais. Objetivo: Avaliar o crescimento cerebelar do nascimento até o termo em recém-nascidos de muito baixo peso (RNMBP). Comparar o diâmetro transverso cerebelar (DTC) de recémnascidos a termo com recém-nascidos de muito baixo peso na idade corrigida correspondente ao termo. Método: RNMBP foram incluídos seqüencialmente no estudo e seus DTCs foram medidos por ultrassonografia craniana nas primeiras 48 horas após o nascimento e no momento da alta hospitalar de todos. A fossa posterior foi examinada através da fontanela Mastóidea usando o aparelho de US GE LOGIC 5 e a medida do DTC realizada no plano coronal. O grupo controle foi formado por recém-nascidos a termo normais com idade gestacional pareada com a idade gestacional pós menstrual corrigida dos RNMBP,sendo seus DTCs medidos nas primeiras 48 horas após o nascimento. Idade gestacional, peso de nascimento, perímetro cefálico e idade da alta foram obtidos dos RNMBP; e idade gestacional, peso de nascimento e perímetro cefálico dos recém-nascidos controles. Teste T de Student’s e correlação de Pearson foram empregados.O estudo foi aprovado pelo Comitê de Ética da instituição. Resultados: Foram incluídos 24 RNMBP e 24 controles. Os RNMBP tiveram idade gestacional media de 30,5± 1,7 semanas, peso de nascimento de 1247± 190 gramas, perímetro cefálico de 27,3 ± 1,5 cm, DTC de 3,28 ± 0,28 cm e idade cronológica da alta de 45,9 ± 12,3 dias. Comparação entre os dois grupos: A idade gestacional corrigida dos RNMBP e a idade gestacional dos controles foram semelhantes (37,04 ± 1,73 semanas e 37,3 ± 1,8 semanas, respectivamente; p= 0,612).O peso dos RNMBP na alta foi significativamente inferior aos controles pareados (2,019 ± 209 g e 2631 ± 423 g; p‹0,0001). Perímetro cefálico dos RNMBP na alta e dos controles, foram respectivamente, 32,23 ± 1,39 centímetros e 33,0 ± 1,6 centímetros (p= 0,098) O DTC aumentou significativamente do nascimento até a alta nos RNMBP; sendo ainda significativamente inferior aos seus pares ao termo ( 4,19 ± 0,41cm e para os controles foi 4,5 ± 0,41 cm; p=0,008). Houve uma correlação positiva entre o perímetro cefálico e o crescimento cerebelar nos RNMBP. Dois RNMBP apresentaram leucomalácia cística e quatro leucomalácia peri-ventricular difusa na ressonância magnética do primeiro ano de vida. Avaliando-se apenas os RNMBP sem leucomalácia peri-ventricular, o crescimento cerebelar foi similar aos recém-nascidos controles (p= 0.135), mas foi menor nos recém-nascidos de muito baixo peso com leucomalácia peri-ventricular quando comparados com os controles (p= 0,005). Conclusões: O crescimento cerebelar foi similar nos recém-nascidos de muito baixo peso sem leucomalácia peri-ventricular e nos controles, e menor naqueles com leucomalácia periventricular em comparação aos controles. Nossos dados sugerem que o crescimento cerebelar é normal na ausência de dano supratentorial. / Introduction: There is little information on cerebellar growth in very low birth weight infants (VLBWI). The cerebellar functions are traditionally reported as tonus, posture and coordination of motricity activity. However, there is an evidence growth of the relation of the cerebellum in cognition, language, memory and motor learning. In VLBWI with neurologic injuries the cerebellum is related to cognitive alterations and emotional disturbance. Objective: Evaluate cerebellar growth from birth up to term in VLBWI. Compare transverse cerebellar diameter (TCD) of term infants and VLBWI at term corrected age. Design/Methods: VLBWI were sequentially included in the study. TCD was measured by cranial ultrasound in the first 48 hours after birth and at hospital discharge in all of them. The posterior fossa was examined through the Mastoid fontanelle using a General Eletric LOGIQ 5 scanner and TCD measurement was taken in the coronal plane. Control group was formed by normal term infants with gestational age that matched VLBWI corrected pos menstrual age. TCD was measured in the first 48 hours after birth. Data on gestational age, birth weight, head circumference, and age at discharge were collected from VLBWI; and gestational age, birth weight, and head circumference from controls newborns. Student’s T test and Pearson correlation were employed. Study was approved by institutional Ethic Committee. Results: We enrolled 24 VLBWI and 24 controls. VLBWI had mean gestational age 30.5±1.7 weeks, birth weight 1247±190 grams, head circumference 27.3 ±1.5cm,TCD 3.28± 0.28 cm, and chronological age at hospital discharge 45.9±12.3 days. Comparison between both groups: VLBWI corrected age at discharge and controls gestational age were similar: 37.4±1.73 weeks and 37.3±1,8 (p=0.612),VLBWI weight at discharge and controls birth weight was significantly inferior to controls: 2019±and 2631±423 grams (p‹ 0.0001),VLBWI head circumference at discharge and controls head circumference: 32.23±1.39 and 33.0 ±1.6 cm (p=0.098),VBWI TCD at discharge and controls TCD: 4.19±0.41 and 4.5±0.41 cm (p=0.008).TCD increased significantly form birth up to hospital discharge in VLBWI (p< 0.001); being still significantly inferior to its term infants matched. There was a significant positive correlation between head circumference and cerebellar growth in VLBWI. Two very low birth weight infants presented cystic and four diffuse peri-ventricular leukomalacia at magnetic resonance image in the first year. Evaluating only the VLBWI without periventricular leukomalacia the cerebellar growth was similar to controls (p=0.135), but it was smaller in VLBWI with peri-ventricular leukomalacia when compared with controls (p=0.005). Hence we divided the case group in with or without peri-ventricular leukomalacia. We compared the two groups with the control group. Conclusions: Cerebellar growth was similar in very low birth Wright infants without periventricular leukomalacia and controls, and smaller in cases with peri-ventricular leukomalacia than in controls. We suggest that cerebelar growth is normal in the absence of supratentorial injury.

The main topic of this thesis are changes in Circadian rhythms caused by cerebellar disorders. Mice with Lurcher mutation, which have specifically degenerated Purkinje cells layer, were choosen as animal model. Our results show that mutation of the glutamate receptor GluRδ2, which causes gradual degeneration of Purkinje cells, leads to damage of Circadian system. Mice with this mutation have reduced capability to adapt to external conditions in different light modes. They are also showing increased variability in endogenous cycle. The mice are also unable to show anticipatory behavior in time-restricted feeding. Compared to control group, affected mice do not show significant rhythm in levels of protein of Bmal1 gene in suprachiasmatic nuclei, paraventricular nuclei nor in habenula. Phosphorylated kinases ERK1/2 and GSK3ß also had distorted rhythms in suprachiasmatic nuclei. Because Circadian oscillations in locomotor activity are partly preserved, Circadian system is likely not damaged on molecular level. Cerebellar mutation hampers synchronization between suprachiasmatic nuclei of neurons and can also affect processes in the ventromedial hypothalamus regulating food intake. Our findings are the first to suggest functional interactions between cerebellum and Circadian pacemaker in suprachiasmatic...

Orientador: Francisco Eduardo Martinez / Coorientador: Marcelo Martinez / Banca: Luiz Fernando Takase / Banca: Daniela Carvalho dos Santos / Banca: Bruno César Schimming / Banca: Cilmery Suemi Kurokaw / Resumo: Introdução: O cerebelo é suscetível aos efeitos lesivos do etanol em exposições agudas ou crônicas, em diferentes idades, podendo ocasionar alterações motoras, de aprendizado, cognitivas e favorecer a morte celular apoptótica. Nosso objetivo foi avaliar a apoptose nos neurônios de Golgi, Purkinje e células granulares do cerebelo de ratos adultos da variedade UChA. Metodologia: Os ratos foram divididos em dois grupos experimentais (20 ratos por grupo): um grupo controle, composto de animais UChA abstinentes e o grupo experimental, formado por ratos UChA, consumidores voluntários de etanol a 10% (˂ 2g etanol/Kg peso corpóreo/dia). Dos 65 dias aos 120 dias de idade, os animais receberam solução de etanol a 10%. Aos 120 dias, os animais foram anestesiados e preparados para eutanásia. O cerebelo foi então analisado por microscopia eletrônica de transmissão (MET), imunoistoquímica para apoptose, caspase-3 (próapoptótica), XIAP (antiapoptótica) e IGF-1R nos neurônios de Golgi, Purkinje e granulares. Avaliou-se também por RT-PCR os genes reguladores da caspase-3, XIAP e IGF-1R no córtex cerebelar dos grupos estudados. Resultados: Os animais UChA mostraram acúmulo de gotas lipídicas, presença de núcleos eletrodensos nos neurônios granulares e de Purkinje, desorganização do corpo medular do cerebelo com alterações na bainha de mielina no córtex cerebelar. Identificou-se também aumento significativo na imunomarcação de caspase-3 e XIAP nos neurônios de Purkinje e granulares. A fragmentação do DNA foi significativa nas células de Purkinje. O IGF-1R não apresentou alterações. Não houve diferenças significativas na expressão dos genes de caspase-3, XIAP, e o IGF-1R. Conclusão: O etanol induz apoptose nas células de Purkinje e granulares do cerebelo de ratos adultos UChA / Abstract: Introduction: The cerebellum is susceptible to the harmful effects of ethanol in acute or chronic exposures at different ages, which may cause motor, learning and cognitive disorders and promote apoptotic cell death (PCD) as well. Our objective was to evaluate apoptosis in Golgi and Purkinje neurons and in granular cell disease of the cerebellum of adult rats of the type UChA. Methodology: The rats were divided in two experimental groups (20 rats per group): a control group, consisting of abstinent UChA animals and the experimental group, formed by UChA rats, volunteers consumers of 10% ethanol (˂ 2g ethanol / kg weight body / day). From 65 day-age to the 120 day-age, the animals received 10% ethanol solution. After 120 days, the rats were anesthetized and prepared for euthanasia. The cerebellum was then analyzed by transmission electron microscopy (TEM), immunohistochemistry to apoptosis, caspase-3 (pro-apoptotic), XIAP (anti-apoptotic) and IGF-1R in Golgi, Purkinje neurons and granular as well. It was also evaluated by RT-PCR (Reverse transcription polymerase chain reaction) the regulatory genes caspase-3, XIAP and IGF-1R in the cerebellar cortex of groups. Results: The UChA animals showed accumulation of lipid droplets, presence of electron-nuclei in granule neurons and Purkinje, disorganization of the medullary body of the cerebellum with some changes in the myelin sheath of the cortex. It was also identified significant increase in the Immunostaining caspase-3 and XIAP in Purkinje neurons and granular. The DNA fragmentation was significant in Purkinje cells. The IGF-1R didn't have any change though. There were no significant differences in the formulation of caspase-3 genes, XIAP and IGF-1R. Conclusion: Ethanol induces apoptosis in granular and Purkinje cells of the cerebellum of adult UChA rats / Doutor

Resumo: Experiências traumáticas na infância estão associadas ao aumento do risco de abuso de álcool e de outras drogas na adolescência e na vida adulta. Crianças e adolescentes maltratados manifestam distúrbios do sistema biológico de resposta ao estresse. A exposição crônica a fatores estressantes aumenta a atividade do eixo hipotálamo-hipófise-adrenal (HHA). Sendo o cerebelo local de ação do etanol, onde o consumo de forma abusiva altera o equilibro e a coordenação motora e que vários aspectos do alcoolismo podem ser frutos do estresse vivido precocemente, este trabalho buscou investigar e avaliar se a separação materna neonatal, aplicada em filhotes machos de ratos UChA e UChB, potencializa os efeitos tóxicos da ingestão crônica de álcool sobre a morfometria dos estratos cerebelares dos animais estudados. Para isso, utilizou-se modelo de alcoolismo experimental (ratos UChA e UChB) e de indução de hiper-reatividade do eixo HHA (Separação Materna). Foram realizadas análises microscópicas e morfométricas. Para morfometria as variáveis estudadas foram: massa corpórea(g), consumo de álcool(g/Kg/dia), altura do córtex cerebelar (μm), altura do estrato molecular(μm), granuloso (μm), purkinjense (μm) e áreas dos citoplasmas das células de Purkinje (μm2). O estresse neonatal não alterou o padrão de consumo de etanol das linhagens UChA e UChB, houve alterações nos níveis plasmáticos de corticoesterona. O córtex cerebelar dos animais UCh mostrou-se menor que no Wistar, sendo a SM fator protetor para atrofia desta região nos grupos SMUChA e SMCO. Os estratos granular e purkinjense mantiveram o padrão de alteração do córtex cerebelar, com a SM atenuando os efeitos do consumo crônico de etanol. Já a camada molecular encontrou-se maior nos animais UChB, onde após indução de hiper-reatividade do eixo HHA ocorreu a atrofia deste grupo. Portanto... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Traumatic experiences in the childhood are associated to an increase in the risk of alcohol and other drugs abuse in the adolescence and in the adulthood. Abused children and adolescents manifest diseases in their biological system in response to that stress. The chronic exposition to stressing factors increases the activity of the hypothalamus-hypophysis-adrenal axis (HHA). The ethanol acts in the cerebellum where the abusive consumption alters the balance and the motor coordination; that is, many aspects of the alcoholism might be consequences of the stress experienced in early years. This research investigated and evaluated if the neonatal maternal separation, applied to UChA and UChB male rats, potentializes the toxic effects of the alcohol chronic ingestion on the cell morphometry of cerebellar extracts in the studied animals. For that, both, the experimental alcoholism model (UChA and UChB rats) and the hipper-reactivity induction of the HHA axis (Maternal Separation) were used. Microscopic and morphometrics analysis were also accomplished. For morphometry, the variables analyzed were: corporal mass (g), alcohol consumption (g/Kg/day), cerebellar cortex height (um), molecular extract height (um), granular (um), Purkinje fibers (um), and cytoplasm areas of the Purkinje cells (um2). The neonatal stress did not alter the ethanol consumption pattern of the UChA and UChB lineages; however, there were alterations in the corticosterone plasmatic levels. The cerebellar cortex of the UCh animals have shown to be smaller than the Wistar ones, and the SM has shown to be a protecting factor for this region atrophy in the SMUChA and SMCO groups. The granular and Purkinje fiber extracts have kept the cerebellar cortex alteration patterns, having the SM lessen the effects of the ethanol chronic consumption. The molecular layer was bigger in the UChB animals, where after the hipperreactivity induction of the HHA axis there was an atrophy of this group. / Orientador: Francisco Eduardo Martinez / Coorientador: Marcelo Martinez / Banca: Luis Fernando Terapelli / Banca: Jair Campos Soares / Mestre

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Hipóxia-isquemia (HI) perinatal é um problema de saúde pública, e ocorrem aproximadamente 1,5 casos de encefalopatias por HI por 1000 nascidos vivos. Dos que sobrevivem 25-60% sofrem de deficiências permanentes do desenvolvimento neurológico, incluindo paralisia cerebral, convulsões, retardo mental, e dificuldade de aprender. Neurônios e oligodendrócitos, especialmente os progenitores, são os mais afetados pela HI. Existem vários modelos de HI, no entanto, poucos levam em consideração as intercorrências maternas, a importância da atividade placentária, e as trocas entre mãe-filho, que são clinicamente observadas em humanos. Robinson estabeleceu um modelo de HI sistêmica pré-natal transitório, onde o fluxo das artérias uterinas da rata grávida era obstruído por 45 minutos no décimo oitavo dia (E18) de gestação. Neste modelo foram observadas alterações que são similares às observadas em cérebros humanos que passaram por hipóxia perinatal, dentre as quais foram relatados aumento no nível de apoptose. Caspase-3 é descrita como uma enzima que atua na apoptose, e é amplamente utilizada como marcador para células apoptóticas. Vários autores vêm mostrando, entretanto, que a enzima caspase-3 pode estar ativada para fins não apoptóticos. No modelo de HI sistêmica pré-natal, foram observados astrogliose na substância branca, morte de oligodendrócitos, lesão em axônios tanto na substância branca como no córtex cerebral, e danos motores. Pouco se sabe da influencia do insulto HI no desenvolvimento do cerebelo, considerando que o cerebelo junto com o córtex motor, contribui para o controle motor. O objetivo desse trabalho foi avaliar a distribuição da caspase-3 clivada durante o desenvolvimento do cerebelo em um modelo de HI pré-natal. Os resultados deste trabalho demonstram que as células caspase-3 clivadas apresentaram duas morfologias distintas em ambos os grupos. Uma onde a caspase-3 foi observada apenas no núcleo, oscilando entre células com imunorreatividade fraca a intensa, e de células com a presença da caspase-3 no corpo celular, nos prolongamentos condensados e presença de fragmentos ao redor do soma, morfologia típica de célula em apoptose. A HI pré-natal, assim como nos hemisférios cerebrais, levou ao aumento de células caspase-3 clivadas com morfologia de progenitores de oligodendrócitos no cerebelo do grupo HI em P2, mas não em P9 e P23. Também foi demonstrado que a HI pré-natal não levou a uma ativação da apoptose em oligodendrócitos, neurônios e microglia (identificados por seus respectivos marcadores, CNPase, NeuN e ED1) apresentando marcação no núcleos de células GFAP+, na substância branca, camada granular e nas células da glia de Bergmann, em P9 e P23 no cerebelo. Podemos concluir que a HI pré-natal aumentou o número de células imunorreativas para a caspase-3 em um período crítico do desenvolvimento da oligodendroglia no cerebelo, e que a diminuição de progenitores de oligodendrócitos no cerebelo decorrente do insulto pré-natal visto em trabalhos anteriores, pode estar relacionada a morte celular por apoptose, embora não se possa descartar a hipótese da participação dessas células que apresentam caspase-3 clivada em outros eventos não apoptóticos desencadeados pela hipóxia-isquemia. / Perinatal hypoxia ischemia (HI) is a public health problem: about 1.5 cases of encephalopathy occurred per 1000 newborn alive. Those who survive 25-60% suffered permanent deficiencies in neurological development, including cerebral palsy seizures, mental retardation and learning difficulties. Neurons and oligodendrocytes, especially the progenitors were the more affected in an HI insult. There are many HI models; however, few of them taken in account the maternal status, the placental activity and the transport of substances between mother and fetus that are clinically observed in humans. Robinson described a model of transitory prenatal systemic HI, in that uterine arteries were clamped for 45 minutes in the eighteenth gestational day (E18). In this model many impairments very similar to those observed in human brain that suffered HI were showed, between them the increase in apoptosis levels. Caspase-3 is related to apoptosis and is largely used as a marker for apoptotic cells. However, many authors have shown that caspase-3 activation may be related not only with apoptosis. In the prenatal systemic HI model were observed white matter astrogliosis, oligodendroglial death, axonal lesion both in white matter as in cerebral cortex as also motor deficits. Little is known about the effects of HI insult in developing cerebellum, which is a region that, together with motor cortex, participates in motor control. The aim of this work was to evaluate cleaved caspase-3 distribution during cerebellar development in a model of prenatal HI. Our results showed that cleaved caspase-3 positive cells showed two different pattern of distribution in both groups. One of them where the caspase-3 was observed only in the nucleus, presenting both low or intense immunoreactivity, and another one presenting the caspase-3 distributed in the cell bodies and in the condensed processes as also the presence of fragments around the soma, a typical morphology of apoptotic cell. Prenatal HI, as also in the cerebral hemispheres leads to an increase in the number of cleaved caspase-3 positive cells that showed oligodendroglial progenitor cell phenotype in the P2 cerebellum, but not in P9 or P23, in HI group. It was also showed that prenatal HI do not leads to Caspase-3 distribution in oligodendrocytes, neurons or microglia (identified by their respective markers, CNPase, NeuN and ED1) however is present in the nucleus of GFAP+ cells, in the white matter, granular layer and in Bergmann Glia at P9 an P23. We may conclude that prenatal HI increased the number of caspase-3 imunorreactive cells in a critical period for oligodendroglial differentiation in developing cerebellum, and that the oligodendroglial progenitors decrease previously showed in the cerebellum as consequence of HI insult may be related to apoptotic cell death, although we may not discard the hypothesis of caspase-3 immunoreactive cells may be related with non apoptotic events triggered by hypoxia-ischemia.