Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 114-137).

No description available.

Thesis (Ph. D. in Neuroscience)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 May 24

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 141 - 176.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Infant human brains show hypomyelination, astrogliosis and impairments in cortical development after perinatal hypoxic-ischemic insults. Robinson and colleagues, using a model of hypoxia-ischemia (HI) in which they clamped the uterine arteries of pregnant rats, showed astrogliosis, oligodendroglial death and axonal rupture in the offspring. In this work we evaluated the expression of 23 cyclic nucleotide 3 phosphodiesterase (CNPase) in motor cortex and cerebellum and the NADPHdiaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this HI model. Rats in the 18th gestation day were anesthetized, the uterine horns were exposed and the four uterine arteries were clamped for 45 minutes. Sham controls had the uterine horns exposed, but no arteries were clamped. Gestation proceeded after surgery. Only full term animals were used. A third group of animals were not submitted to the surgical procedure (NM). Animals were anesthetized and perfused with 4% paraformaldehyde at 9, 23 and 90 days. Coronal prosencephalus and parassagital cerebellum sections were submitted to CNPase immunohistochemistry. Additionally, parassagital cerebellum sections were submitted to NADPH-d hystochemistry. We observed a decrease in CNPase immunoreactivity in the cingulum of HI animals at all ages compared with NM and Sham animals. At P9 a lower number of CNPase+ cells in the cingulum and motor cortex of HI animals was observed. At P23 we observed a lower number of CNPase+ cells and fibers in the motor cortex and a greater number of cells in the cingulum of HI animals. A lower number of CNPase+ fibers were observed in the HI animals in the motor cortex at P90. In the cerebellum, at P9, CNPase+ fibers and cells were observed in more distal positions in the folium 1 of Sham and NM than in HI animals. The immunoreactivity to CNPase was altered in the white matter and in the fibers in the gray matter of HI animals at P23 and P90. The cerebelar white matter of HI animals showed a greater number of NADPH-d+ cells than that of control groups at both P9 and P23. At P23 the labeling was observed in the body as well as in the processes. Purkinje cells of Sham animals showed NADPH-d+ labeling in the body and dendritic arborization at folium 1, meanwhile animals of HI group did not show this labeling in the body, but the dendritic arborization had a widespread strong labeling. HI animals at P23 showed NADPHd+ cells in the molecular layer of cerebellum folia 1, 6 and 10. Sham or NM animals did not show NADPH-d+ cell in this region. This pattern was maintained up to P90. Our results showed impairments in oligodendroglial cells that may have been caused by reductions in the number of progenitors, alterations in migration patterns and/or delays in differentiation. We also showed that perinatal HI altered the distribution of NADPH-d+ cells during cerebelar development and that some alterations persist into adulthood, which suggests a permanent impairment. These alterations could lead to motor deficits in young adult HI rats, which, if present, could be used in devising new therapeutic strategies. / Crianças que sofrem insultos hipóxico-isquêmicos perinatais têm hipomielinização, astrogliose e desenvolvimento cortical alterado. Robinson e cols., usando um modelo de hipóxia-isquemia (HI) por obstrução das artérias uterinas na rata grávida, mostraram astrogliose, morte de oligodendrócitos e ruptura axonal na prole. Neste trabalho avaliamos a expressão de 23nucleotídeo 3fosfodiesterase (CNPase) no córtex e cerebelo e a distribuição da NADPH-diaforase (NADPH-d) no cerebelo de ratos submetidos a este modelo de HI. Ratas no 18 dia de gestação foram anestesiadas, os cornos uterinos expostos e as 4 artérias uterinas obstruídas por 45 minutos (Grupo HI). Animais controle tiveram os úteros expostos sem sofrer a obstrução (Grupo Sham). Após a cirurgia a gestação prosseguiu. Somente animais nascidos a termo foram utilizados. Um terceiro grupo de animais não foi operado (Grupo NM). Após anestesia e perfusão-fixação com paraformaldeído 4% aos 9, 23 e 90 dias pós-natais, cortes coronais do prosencéfalo e parassagitais do cerebelo foram obtidos em criótomo e submetidos à imunohistoquímica para CNPase. Adicionalmente, cortes parassagitais do cerebelo obtidos em criótomo foram submetidos à histoquímica para NADPH-d. Observamos uma diminuição da imunorreatividade à CNPase no cingulum dos animais HI comparados aos animais NM e Sham em todas as idades. Em P9, nos animais HI, há menor número de células CNPase+ no cingulum e córtex motor. Aos 23 dias, há um menor número de células e fibras CNPase+ no córtex motor e mais corpos celulares no cingulum dos animais HI. Aos 90 dias, os animais HI apresentaram menos fibras no córtex motor que os animais controle. No cerebelo, observamos corpos celulares e fibras CNPase+ em regiões mais distais na folha 1 nos animais NM e Sham que nos animais HI aos 9 dias. A imunorreatividade à CNPase estava alterada na substância branca e nas fibras na substância cinzenta nos animais HI tanto aos 23 quanto aos 90 dias. A substância branca nos animais HI apresentaram maior numero de células marcadas para NADPH-d que os animais controle aos 9 e 23 dias, sendo que em P23 a marcação se encontrava tanto no corpo celular quanto nos prolongamentos. As células de Purkinje na folha 1 apresentaram marcação para NADPH-d+, tanto no corpo celular quanto na árvore dendrítica, nos animais NM e Sham, enquanto nos animais HI somente a árvore dendrítica apresentava marcação. Animais do grupo HI aos 23 dias apresentaram células NADPH-d+ na camada molecular das folhas 1, 6 e 10, sendo que animais dos grupos controle não apresentaram células NADPH-d+ nesta região. Este padrão de marcação se manteve nos animais adultos (P90). Nossos resultados mostram alterações na oligodendroglia que podem ser causadas por um menor número de células progenitoras, problemas na migração aos destinos finais e/ou atraso na diferenciação. Mostramos também que a HI perinatal altera a distribuição das células NADPH-d+ durante o desenvolvimento do cerebelo, com algumas destas alterações mantidas na vida adulta, indicando um dano permanente nesta estrutura. Estas alterações poderão resultar em distúrbios motores nos ratos adultos jovens que sofrerem HI perinatal, constituindo então um modelo interessante para desenvolver novas estratégias terapêuticas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Infant human brains show hypomyelination, astrogliosis and impairments in cortical development after perinatal hypoxic-ischemic insults. Robinson and colleagues, using a model of hypoxia-ischemia (HI) in which they clamped the uterine arteries of pregnant rats, showed astrogliosis, oligodendroglial death and axonal rupture in the offspring. In this work we evaluated the expression of 23 cyclic nucleotide 3 phosphodiesterase (CNPase) in motor cortex and cerebellum and the NADPHdiaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this HI model. Rats in the 18th gestation day were anesthetized, the uterine horns were exposed and the four uterine arteries were clamped for 45 minutes. Sham controls had the uterine horns exposed, but no arteries were clamped. Gestation proceeded after surgery. Only full term animals were used. A third group of animals were not submitted to the surgical procedure (NM). Animals were anesthetized and perfused with 4% paraformaldehyde at 9, 23 and 90 days. Coronal prosencephalus and parassagital cerebellum sections were submitted to CNPase immunohistochemistry. Additionally, parassagital cerebellum sections were submitted to NADPH-d hystochemistry. We observed a decrease in CNPase immunoreactivity in the cingulum of HI animals at all ages compared with NM and Sham animals. At P9 a lower number of CNPase+ cells in the cingulum and motor cortex of HI animals was observed. At P23 we observed a lower number of CNPase+ cells and fibers in the motor cortex and a greater number of cells in the cingulum of HI animals. A lower number of CNPase+ fibers were observed in the HI animals in the motor cortex at P90. In the cerebellum, at P9, CNPase+ fibers and cells were observed in more distal positions in the folium 1 of Sham and NM than in HI animals. The immunoreactivity to CNPase was altered in the white matter and in the fibers in the gray matter of HI animals at P23 and P90. The cerebelar white matter of HI animals showed a greater number of NADPH-d+ cells than that of control groups at both P9 and P23. At P23 the labeling was observed in the body as well as in the processes. Purkinje cells of Sham animals showed NADPH-d+ labeling in the body and dendritic arborization at folium 1, meanwhile animals of HI group did not show this labeling in the body, but the dendritic arborization had a widespread strong labeling. HI animals at P23 showed NADPHd+ cells in the molecular layer of cerebellum folia 1, 6 and 10. Sham or NM animals did not show NADPH-d+ cell in this region. This pattern was maintained up to P90. Our results showed impairments in oligodendroglial cells that may have been caused by reductions in the number of progenitors, alterations in migration patterns and/or delays in differentiation. We also showed that perinatal HI altered the distribution of NADPH-d+ cells during cerebelar development and that some alterations persist into adulthood, which suggests a permanent impairment. These alterations could lead to motor deficits in young adult HI rats, which, if present, could be used in devising new therapeutic strategies. / Crianças que sofrem insultos hipóxico-isquêmicos perinatais têm hipomielinização, astrogliose e desenvolvimento cortical alterado. Robinson e cols., usando um modelo de hipóxia-isquemia (HI) por obstrução das artérias uterinas na rata grávida, mostraram astrogliose, morte de oligodendrócitos e ruptura axonal na prole. Neste trabalho avaliamos a expressão de 23nucleotídeo 3fosfodiesterase (CNPase) no córtex e cerebelo e a distribuição da NADPH-diaforase (NADPH-d) no cerebelo de ratos submetidos a este modelo de HI. Ratas no 18 dia de gestação foram anestesiadas, os cornos uterinos expostos e as 4 artérias uterinas obstruídas por 45 minutos (Grupo HI). Animais controle tiveram os úteros expostos sem sofrer a obstrução (Grupo Sham). Após a cirurgia a gestação prosseguiu. Somente animais nascidos a termo foram utilizados. Um terceiro grupo de animais não foi operado (Grupo NM). Após anestesia e perfusão-fixação com paraformaldeído 4% aos 9, 23 e 90 dias pós-natais, cortes coronais do prosencéfalo e parassagitais do cerebelo foram obtidos em criótomo e submetidos à imunohistoquímica para CNPase. Adicionalmente, cortes parassagitais do cerebelo obtidos em criótomo foram submetidos à histoquímica para NADPH-d. Observamos uma diminuição da imunorreatividade à CNPase no cingulum dos animais HI comparados aos animais NM e Sham em todas as idades. Em P9, nos animais HI, há menor número de células CNPase+ no cingulum e córtex motor. Aos 23 dias, há um menor número de células e fibras CNPase+ no córtex motor e mais corpos celulares no cingulum dos animais HI. Aos 90 dias, os animais HI apresentaram menos fibras no córtex motor que os animais controle. No cerebelo, observamos corpos celulares e fibras CNPase+ em regiões mais distais na folha 1 nos animais NM e Sham que nos animais HI aos 9 dias. A imunorreatividade à CNPase estava alterada na substância branca e nas fibras na substância cinzenta nos animais HI tanto aos 23 quanto aos 90 dias. A substância branca nos animais HI apresentaram maior numero de células marcadas para NADPH-d que os animais controle aos 9 e 23 dias, sendo que em P23 a marcação se encontrava tanto no corpo celular quanto nos prolongamentos. As células de Purkinje na folha 1 apresentaram marcação para NADPH-d+, tanto no corpo celular quanto na árvore dendrítica, nos animais NM e Sham, enquanto nos animais HI somente a árvore dendrítica apresentava marcação. Animais do grupo HI aos 23 dias apresentaram células NADPH-d+ na camada molecular das folhas 1, 6 e 10, sendo que animais dos grupos controle não apresentaram células NADPH-d+ nesta região. Este padrão de marcação se manteve nos animais adultos (P90). Nossos resultados mostram alterações na oligodendroglia que podem ser causadas por um menor número de células progenitoras, problemas na migração aos destinos finais e/ou atraso na diferenciação. Mostramos também que a HI perinatal altera a distribuição das células NADPH-d+ durante o desenvolvimento do cerebelo, com algumas destas alterações mantidas na vida adulta, indicando um dano permanente nesta estrutura. Estas alterações poderão resultar em distúrbios motores nos ratos adultos jovens que sofrerem HI perinatal, constituindo então um modelo interessante para desenvolver novas estratégias terapêuticas.

Yang, Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 222-244). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Awards and Presentations --- p.ii / Abstract in English --- p.iii / Abstract in Chinese --- p.vii / Table of Contents --- p.x / List of Tables --- p.xv / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Gliomas --- p.1 / Chapter 1.1.1 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.1.2 --- Glioblastoma multiforme (GBM) --- p.3 / Chapter 1.1.3 --- Molecular pathology of gliomas --- p.4 / Chapter 1.1.3.1 --- Genetic alterations in OTs --- p.4 / Chapter 1.1.3.2 --- Prognostic and predictive factors in OTs --- p.7 / Chapter 1.1.3.3 --- Genetic alterations in GBM --- p.8 / Chapter 1.1.3.4 --- Prognostic and predictive factors in GBM --- p.10 / Chapter 1.2 --- microRNA(miRNA) --- p.13 / Chapter 1.2.1 --- miRNA biogenesis and function --- p.13 / Chapter 1.2.2 --- miRNA involvement in cancer --- p.17 / Chapter 1.2.2.1 --- Dysregulation of miRNAs in human malignancies --- p.17 / Chapter 1.2.2.2 --- Function and potential application of miRNAs --- p.17 / Chapter 1.2.3 --- Role of miRNAs in glioma --- p.19 / Chapter 1.2.3.1 --- miRNAs in OTs --- p.19 / Chapter 1.2.3.2 --- miRNAs in GBM --- p.20 / Chapter 1.3 --- miR-137 --- p.30 / Chapter 1.3.1 --- Biology of miR-137 --- p.30 / Chapter 1.3.2 --- Role of miR-137 in carcinogenesis --- p.33 / Chapter 1.3.2.1 --- Deregulation of miR-137 in cancer --- p.33 / Chapter 1.3.2.2 --- Regulation of miR-137 expression in cancer --- p.33 / Chapter 1.3.2.3 --- Biological functions of miR-137 in cancer --- p.37 / Chapter 1.3.3 --- Role of miR-137 in differentiation and neurogenesis --- p.39 / Chapter CHAPTER 2 --- AIMS OF STUDY --- p.43 / Chapter CHARPTER 3 --- MATERIALS AND METHODS --- p.45 / Chapter 3.1 --- Tumor samples --- p.45 / Chapter 3.2 --- Cell lines and culture conditions --- p.48 / Chapter 3.3 --- Fluorescence in situ hybridization (FISH) --- p.49 / Chapter 3.4 --- Cell transfection --- p.52 / Chapter 3.4.1 --- Transfection of oligonucleotides --- p.52 / Chapter 3.4.1.1 --- Oligonucleotide preparation --- p.52 / Chapter 3.4.1.2 --- Optimization of transfection condition --- p.52 / Chapter 3.4.2 --- Cotransfection of plasmids and miRNA mimic --- p.53 / Chapter 3.4.2.1 --- Optimization of transfection condition --- p.53 / Chapter 3.4.2.2 --- Procedure of transfection --- p.54 / Chapter 3.5 --- Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) --- p.55 / Chapter 3.5.1 --- RNA extraction from frozen tissues and cell lines --- p.55 / Chapter 3.5.2 --- qRT-PCR for miR-137 --- p.56 / Chapter 3.5.3 --- qRT-PCR for CSE1L and ERBB4 transcripts --- p.57 / Chapter 3.6 --- 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) treatment --- p.61 / Chapter 3.7 --- Western blotting --- p.62 / Chapter 3.7.1 --- Preparation of cell lysate --- p.62 / Chapter 3.7.2 --- Measurement of protein concentration --- p.62 / Chapter 3.7.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.63 / Chapter 3.7.4 --- Electroblotting of proteins --- p.67 / Chapter 3.7.5 --- Immunoblotting --- p.67 / Chapter 3.8 --- Dual-luciferase reporter assay --- p.70 / Chapter 3.8.1 --- Construction of reporter plasmids --- p.70 / Chapter 3.8.1.1 --- Experimental outline --- p.70 / Chapter 3.8.1.2 --- PCR Amplification of MREs --- p.70 / Chapter 3.8.1.3 --- TA cloning --- p.71 / Chapter 3.8.1.4 --- Transformation --- p.72 / Chapter 3.8.1.5 --- Blue/white screening and validation of recombinants --- p.72 / Chapter 3.8.1.6 --- Subcloning of 3'UTR fragments into pMIR-reproter vector --- p.73 / Chapter 3.8.2 --- Site-directed mutagenesis --- p.74 / Chapter 3.8.3 --- Plasmid and miRNA mimic cotransfection --- p.76 / Chapter 3.8.4 --- Determination of luciferase activity --- p.76 / Chapter 3.9 --- Functional assays : --- p.79 / Chapter 3.9.1 --- Cell growth and proliferation assay --- p.79 / Chapter 3.9.1.1 --- "3-(4，5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.79 / Chapter 3.9.1.2 --- Cell counting --- p.80 / Chapter 3.9.1.3 --- 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay --- p.80 / Chapter 3.9.2 --- Apoptosis assay --- p.82 / Chapter 3.9.3 --- Anchorage-independent growth assay --- p.82 / Chapter 3.9.4 --- Wound healing assay --- p.83 / Chapter 3.9.5 --- Matrigel invasion assay --- p.84 / Chapter 3.9.6 --- Cell differentiation assay --- p.85 / Chapter 3.10 --- Immunohistochemical analysis --- p.86 / Chapter 3.10.1 --- H&E staining --- p.86 / Chapter 3.10.2 --- Detection of Ki-67 expression --- p.87 / Chapter 3.10.3 --- Detection of CSE1L expression --- p.87 / Chapter 3.10.4 --- Scoring methods --- p.88 / Chapter 3.11 --- Bioinformatic analysis --- p.90 / Chapter 3.12 --- Statistical analysis --- p.92 / Chapter CHAPTER 4 --- RESULTS --- p.93 / Chapter 4.1 --- Expression of miR-137 in glioma cells and clinical significance --- p.93 / Chapter 4.1.1 --- Description of 36 OT samples --- p.93 / Chapter 4.1.2 --- miR-137 level in oligodendroglial tumors and glioma cells --- p.102 / Chapter 4.1.3 --- "Association of miR-137 expression with clinicopathological features, lp/19q status and Ki-67 expression" --- p.104 / Chapter 4.2 --- miR-137 levels in glioma cells after demethylation treatment --- p.113 / Chapter 4.3 --- Biological effects of miR-137 overexpression in glioma cells --- p.118 / Chapter 4.3.1 --- Cell growth --- p.118 / Chapter 4.3.1.1 --- Cell viability --- p.118 / Chapter 4.3.1.2 --- Cell number --- p.123 / Chapter 4.3.1.3 --- Cell cycle analysis : --- p.127 / Chapter 4.3.2 --- Anchorage-independent cell growth --- p.130 / Chapter 4.3.3 --- Cell apoptosis --- p.134 / Chapter 4.3.4 --- Cell motility --- p.136 / Chapter 4.3.5 --- Cell differentiation : --- p.142 / Chapter 4.4 --- Identification of miR-137 targets --- p.144 / Chapter 4.4.1 --- In silico prediction of potential miR-137 targets --- p.144 / Chapter 4.4.2 --- Experimental validation of miR-137 targets by dual-luciferase reporter assay --- p.147 / Chapter 4.4.3 --- "Expression of miR-137 candidate targets, CSE1L and ERBB4 in glioma cells" --- p.152 / Chapter 4.4.4 --- Effects of miR-137 on CSE1L transcript and protein levels --- p.154 / Chapter 4.5 --- Expression of CSE1L in OTs --- p.156 / Chapter 4.5.1 --- CSE1L expression in OTs by qRT-PCR and IHC --- p.156 / Chapter 4.5.2 --- Correlation of CSE1L expression with clinicopathological features --- p.165 / Chapter 4.6 --- Effects of CSE1L knockdown in glioma cells --- p.168 / Chapter 4.6.1 --- Cell growth --- p.170 / Chapter 4.6.1.1 --- Cell viability --- p.170 / Chapter 4.6.1.2 --- Cell number --- p.173 / Chapter 4.6.1.3 --- Cell cycle analysis --- p.176 / Chapter 4.6.2 --- Anchorage-independent cell growth --- p.179 / Chapter 4.6.3 --- Cell apoptosis --- p.182 / Chapter 4.6.4 --- Cell motility --- p.184 / Chapter CHAPTER 5 --- DISCUSSION --- p.190 / Chapter 5.1 --- Expression of miR-137 transcript level in OTs and glioma cell lines --- p.190 / Chapter 5.2 --- Association of miR-137 expression with OT clinical and molecular parameters --- p.192 / Chapter 5.3 --- Prognostic significance of clinical features and miR-137 expression in OTs --- p.194 / Chapter 5.4 --- Inactivation mechanisms of miR-137 in glioma --- p.196 / Chapter 5.5 --- Biological effects of miR-137 overexpression in glioma cells --- p.198 / Chapter 5.6 --- CSE1L is a novel miR-137 target in glioma --- p.200 / Chapter 5.7 --- Expression of CSE1L in glioma --- p.203 / Chapter 5.8 --- Intracellular distribution of CSElL in OTs --- p.206 / Chapter 5.9 --- Correlation of CSE1L expression with clinicopathological and molecular features in OTs --- p.208 / Chapter 5.10 --- CSE1L mediates effects of miR-137 in glioma cells --- p.210 / Chapter 5.11 --- Biological roles of CSE1L in glioma cells 226}0Ø. --- p.212 / Chapter 5.11.1 --- CSE1L in glioma cell proliferation --- p.212 / Chapter 5.11.2 --- CSE1L in glioma cell apoptosis --- p.213 / Chapter 5.11.3 --- CSE1L in glioma cell invasion --- p.215 / Chapter CHAPTER 6 --- CONCLUSIONS --- p.216 / Chapter CHAPTER 7 --- FUTURE STUDIES --- p.219 / Chapter 7.1 --- Expression Molecular mechanisms for miR-137 inactivation in glioma --- p.219 / Chapter 7.2 --- Identification of more miR-137 targets in glioma --- p.219 / Chapter 7.3 --- Role of miR-137 and CSE1L in drug-induced apoptosis in glioma --- p.220 / Chapter 7.4 --- Deciphering dysregulated and clinical relevant miRNAs in glioma --- p.220 / Chapter 7.5 --- Effects of miR-137 in vivo and the therapeutic potential in glioma treatment --- p.221 / REFERENCES --- p.222

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.

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.