Os autores agradecem a Dan Firl, Chris Corbett e Jesse Denson, PhD. O financiamento foi fornecido pelo NIH R01 NINDS NS060765 para SR, o P30 Cobre Programa Piloto para a LJ e do Programa de Saúde da Criança Assinatura para LJ na Universidade do Novo México.

Cuidado Institucional e Comissões Use no hospital de ambos Boston Children ea Universidade do Novo México Centro de Ciências da Saúde aprovou todos os procedimentos experimentais. NOTA: Antes de iniciar o procedimento, selo, esterilizar e autoclave todos os instrumentos cirúrgicos e campos cirúrgicos. Além disso, preparar medicamentos pós-operatórios em frascos esterilizados, incluindo 0,125% bipivucaine e 0,1 mg / kg de buprenorfina. Também preparar a solução de lipopolissacarídeo (LPS) de forma estéril: 0,04 mg / ml de LPS (0111: B4) em solução salina estéril contendo diluir corante azul de Evan. 1. Anestesia <ol><li> Induzir a anestesia em um dia embrionário 18 (E18) grávida de ratos Sprague Dawley com uma mistura de 3% de isoflurano equilibrado de nitrogênio 70% e 30% de oxigênio. </li><li> Remover rato a partir da câmara de indução e colocar o rato na posição supina drapeado cirúrgica cobertor de circulação de água regulado a 37 ° C. Transferir para anestesia nariz cone e reduzir isofluranível ne a 2%. </li><li> Gentilmente aplicar pomada oftálmica a cada olho para evitar o ressecamento da córnea. Durante o procedimento de monitorar continuamente a temperatura, taxa de respiração e batimentos cardíacos do animal. Fisiologia materna deveria permanecer estável durante todo o procedimento. </li></ol> 2. Prep Cirúrgica e Scrub <ol><li> Usando pequenas tesouras remover todos os pêlos na região abdominal inferior. Barbear em um padrão retangular com cuidado para evitar entalhar os mamilos ou a geração de erupção de barbear que pode ser irritante para o futuro dos cuidados de filhotes nascidos vivos. </li><li> Prepare a pele abdominal através da aplicação de iodopovidona e 70% de etanol matagal alternando com cotonetes estéreis. Repita o matagal tal que iodopovidona e 70% de etanol são cada aplicada 3x de maneira alternada. Deixe secar. </li><li> Confirme profundidade da anestesia via ausência de toe-pitada reflexo. Na ausência do reflexo à dor e estímulos, reduzir o nível de isoflurano a 1%. </li><li> Usando toalhas cirúrgicos estéreis,armar o animal. Tenha cuidado para colocar as cortinas em um ângulo adequado de tal forma que eles maximizar a quantidade de fluido de irrigação que absorvem enquanto não obstruindo o fluxo sanguíneo para os cornos uterinos. </li></ol> 3. Abdominal Laparotomia <ol><li> Usando um bisturi de fazer uma incisão na linha média de 3 cm de pele abdominal preparado. Sem rodeios dissecar a camada de pele da fáscia abdominal com uma tesoura. Usando fórceps e tesouras cirúrgicas, elevar a camada fascial abdominal e fazer uma incisão da linea alba avascular a ganhar acesso à cavidade peritoneal. </li><li> Coloque gaze cirúrgica no exterior da incisão e humedecer com solução salina estéril. Utilizando uma pinça sem corte e pressão externa sobre o abdômen, remova cuidadosamente os cornos uterinos da cavidade peritoneal e organizar na gaze umedecida. </li><li> Evitar cuidadosamente emaranhamento e entre em contato com o intestino. Organizar fetos usando uma pinça, contactando somente o tecido muscular entre sacos amniótico individuais.Expor e isolar as artérias uterinas 4 utilizando dissecção romba. NOTA: É preciso ter cuidado para dissecar as artérias uterinas. Tecido circundante e navios próprios são extremamente delicada. Danos aos vasos maternos pode causar sangramento e, em casos graves, morte fetal e materna. </li></ol> 4. Colocação de aneurisma Clipes <ol><li> Coloque um clipe de aneurisma rato G 30 em cada artéria uterina. Assegurar a cessação do fluxo sanguíneo, incluindo proximais e distais impulsos, e escurecimento dos vasos uterinos incluindo placentas individuais. Cubra as pontas expostas e toda campo cirúrgico com gaze e irrigar com solução salina estéril. Tome cuidado para manter o campo úmido com irrigação aproximadamente a cada 10 min. </li><li> Depois de 60 minutos, retire a gaze e irrigar o campo. Certifique-se de que os cornos uterinos e as embarcações estão devidamente umedecido para remoção clipe bem sucedido. Remova cuidadosamente cada clipe de aneurisma com a pinça. Tome cuidado para não causar trauma para o navio, e maintintegridade do tecido ain durante a remoção. </li><li> Completamente irrigar os cornos uterinos e campo, tendo o cuidado de remover quaisquer fios soltos de gaze dos sacos amniótico. </li></ol> 5. A injeção de lipopolissacarídeo para amniótico Sacs <ol><li> Na base de cada indivíduo saco amniótico, imediatamente anterior à placa placentária, injectar 100 ul de LPS (4 mg / SAC) com corante azul de Evan em diluída para o fluido amniótico. Use uma pinça sem corte para estabilizar e girar cada saco amniótico para uma posição ideal para injecção. Corante azul de Evan diluído é um agente de contraste que é útil para confirmar a colocação de seringa adequada e injeção. NOTA: Use apenas um ultra-fino seringa de 0,3 ml de insulina com anexado 8 milímetros 31 G agulha para as injeções intra-amniótica. Usando agulhas de calibre maiores resultará na perda de líquido amniótico crônica, morte fetal e reabsorção da gravidez. Pequenas quantidades de fuga de líquido amniótico após a remoção da seringa pode ser mitigciado por pressão direta para o saco amniótico. Alguns fetos de ratos pode tolerar um grau de oligohydramnios. No entanto, a perda de líquido amniótico aguda de punção com agulhas de calibre grandes, ou punção acidental, resultando em vazamento de fluido crônica, resulta em perda fetal e, em casos graves, perda de gravidezes vizinhos. </li><li> Irrigar o cornos uterinos 3x com solução salina estéril. </li></ol> 6. Fechando a Laparotomia <ol><li> Utilizando uma pinça, voltar cuidadosamente os cornos uterinos para a cavidade peritoneal. Garantindo um espaço adequado entre os sacos amniótico ea incisão na linha média, re-aproximar as bordas da camada musculofascial usando uma sutura de seda 3-0 em execução. Esteja ciente da colocação dos sacos amniótico ao fechar a incisão muscular. Tenha cuidado para não suturar em ou através de um saco. </li><li> Re-aproximar a camada de pele, utilizando uma sutura de seda 3-0 execução, o fechamento da camada de pele. NOTA: A laparotomia deve ser fechada em duas camadas de sutura contínua para permitirpara a pele e expansão muscular com o aumento da gestação. Suturas contínuas permitir a tensão ferida uniformemente distribuída. Suturas interrompidas são menos desejado como vários nós são irritantes e podem ser facilmente mastigado pelo rato em cima de recuperação da anestesia. Grampos cirúrgicos não são desejados. Caudas dos nós cirúrgicos deve ser cortado muito curto (&lt;3 mm). </li><li> Injectar 1 mL de 0.125% de bupivacaína por via subcutânea em torno de bordas da ferida usando uma agulha 26 G. Administrar uma dose de 0,1 mg / kg de buprenorfina por via subcutânea na nuca do pescoço. </li><li> Desligue o isoflurano e toalha rato seco, se necessário. Coloque na gaiola casa limpa e monitorar a recuperação da anestesia. Certifique-rato não se torne hipotérmica. </li></ol> 7. Recuperação Pós-Operatória e Cuidados <ol><li> Monitorar o rato cada 8-12 horas para 72 horas e em seguida diariamente até filhotes nascem (aproximadamente E22 ou E23). Administrar doses adicionais de buprenorfina q8-12 h / 72 h ou prn como ditado pelo IACUC. </li><li> Monitorar ratos para sinais de dor, desconforto, sangramento vaginal ou sangramento do sítio cirúrgico. Inspecionar as suturas e incisão e tomar cuidado para garantir rato não está mastigando ou remover suas suturas prematuramente. Embora excepcionalmente raro, os ratos que comprometem suas suturas estão em risco de deiscência da ferida. NOTA: Ocasionalmente ratos podem ingerir quantidades excessivas de cama ou não alimentares, denominado pica, como um efeito colateral da administração buprenorfina. Embora muito raro, os ratos devem ser monitorados para pica e obstrução do intestino potencial subseqüente. </li></ol> 8. Tissue Processing e cryosectioning <ol><li> Para se preparar para Hematoxilina e Eosina (H &amp; E) coloração do cérebro pós-natal, remover os filhotes de sua gaiola de origem no dia 2 pós-natal (P2). Usando uma tesoura cirúrgica decapitar filhotes de ratos e retire cuidadosamente o cérebro do crânio. </li><li> Gota correcção cérebro em um tubo de 15 ml contendo 7 ml de paraformaldeído a 4% em solução salina tamponada com fosfato(PBS). Coloque cérebro a 4 ° C e fixar durante 72 h. </li><li> Após 72 horas, transferir os cérebros a uma solução de PBS estéril contendo 30% de sacarose (w / v) e voltar a 4 ° C. NOTA: Uma vez que os cérebros cair na solução de sacarose eles estão prontos para ser seccionado em um criostato. </li><li> Rapidamente congelar cérebros e montar no pilão cryostat para aquisição de cortes coronais congeladas. Cortar 20 mm cortes coronais congeladas e montagem em lâminas. Garantir seções são recolhidas em série. </li><li> Permitir que as lâminas secar à temperatura ambiente durante a noite. Loja desliza a -20 ° C. </li></ol> 9. Hematoxilina &amp; eosina <ol><li> Tome slides montado cortes congelados e quente à temperatura ambiente. NOTA: Prepare todas as soluções fresco. </li><li> Colocar as lâminas em conjunto uma lâmina de aquecedor para 50 ° C durante 2 h. </li><li> Transferir as lâminas para um suporte de coloração. Dip desliza 10x em água deionizada bidestilada (ddH2O). </li><li> Incubar as lâminas em hematoxilina 100% para 5 min. Time em hematoxilina pode ser otimizada dependendo do grau de coloração roxa. </li><li> Dip slides em 4x torneira H 2 O, e deixe descansar em limpo torneira H2O para 1 min. </li><li> Dip slides para 15x ácido em álcool (250 ml etanol a 70% + 1 ml de ácido clorídrico concentrado). </li><li> Dip slides em 4x torneira H 2 O, e deixe descansar em limpo torneira H2O para 1 min. </li><li> Incubar em 1% de carbonato de lítio durante 2 min. </li><li> Dip slides em 4x torneira H 2 O, e deixe descansar em limpo torneira H2O para 1 min. </li><li> Incubar em etanol a 95% durante 1 min. </li><li> Dip slides 7x em 100% Eosina. Número de mergulhos em Eosina pode ser modificado dependendo do grau de coloração rosa. </li><li> Dip slides 5x em etanol a 95%. </li><li> 5x Dip slides em uma nova mudança de etanol a 95%. </li><li> Incubar as lâminas em etanol a 100% durante 1 min. </li><li> Incubar as lâminas em uma nova mudança de etanol a 100% durante 1 min. </li><li> Incubar as lâminas em 100% de xileno para 15 min. NOTA: Os passos xileno elamínulas deve ocorrer em um exaustor. </li><li> Incubar as lâminas em novas mudanças de xileno para 15 min. </li><li> Lamela com Permount e deixe secar no exaustor. </li><li> Imagem slides com um microscópio de luz. </li></ol>

<ol>
	<li>Blencowe, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+birth+associated+neurodevelopmental+impairment+estimates+at+regional+and+global+levels+for+2010.">Preterm birth associated neurodevelopmental impairment estimates at regional and global levels for 2010.</a> Pediatr Res. 74, 17-34 (2013).</li><li>Mwaniki, M. K., Atieno, M., Lawn, J. E., Newton, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+neurodevelopmental+outcomes+after+intrauterine+and+neonatal+insults+a+systematic+review.">Long term neurodevelopmental outcomes after intrauterine and neonatal insults a systematic review.</a> Lancet. 379, 445-452 (2012).</li><li>Dammann, O., Leviton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+or+sustained+systemic+inflammation+and+the+preterm+brain.">Intermittent or sustained systemic inflammation and the preterm brain.</a> Pediatr Res. 75, 376-380 (2014).</li><li>Leviton, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiologic+and+histologic+characteristics+of+the+extremely+preterm+infant's+placenta+predict+white+matter+damage+and+later+cerebral+palsy.">Microbiologic and histologic characteristics of the extremely preterm infant's placenta predict white matter damage and later cerebral palsy.</a> the ELGAN study Pediatr Res. 67, 95-101 (2010).</li><li>Redline, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+responses+in+the+placenta+and+umbilical+cord.">Inflammatory responses in the placenta and umbilical cord.</a> Semin Fetal Neonatal Med. 11, 296-301 (2006).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+chorioamnionitis+is+the+most+common+placental+lesion+in+late+preterm+birth.">Chronic chorioamnionitis is the most common placental lesion in late preterm birth.</a> Placenta. 34, 681-689 (2013).</li><li>Lee, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+histologic+chorioamnionitis+is+a+risk+factor+for+adverse+neonatal+outcome+in+late+preterm+birth+after+preterm+premature+rupture+of+membranes.">Acute histologic chorioamnionitis is a risk factor for adverse neonatal outcome in late preterm birth after preterm premature rupture of membranes.</a> PloS one. 8, e79941 (2013).</li><li>Pappas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chorioamnionitis+and+early+childhood+outcomes+among+extremely+low+gestational+age+neonates.">Chorioamnionitis and early childhood outcomes among extremely low gestational age neonates.</a> JAMA Peds. 168, 137-147 (2014).</li><li>Gaillard, R., Arends, L. R., Steegers, E. A., Hofman, A., Jaddoe, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+and+third+trimester+placental+hemodynamics+and+the+risks+of+pregnancy+complications+the+Generation+R+Study.">Second and third trimester placental hemodynamics and the risks of pregnancy complications the Generation R Study.</a> Am J Epidemiol. 177, 743-754 (2013).</li><li>Trivedi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+placental+inflammation+but+not+adrenal+activation+is+associated+with+extreme+preterm+delivery.">Fetal placental inflammation but not adrenal activation is associated with extreme preterm delivery.</a> Am J Obstet Gynecol. 206, 236 (2012).</li><li>Yanowitz, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+disturbances+in+premature+infants+born+after+chorioamnionitis+association+with+cord+blood+cytokine+concentrations.">Hemodynamic disturbances in premature infants born after chorioamnionitis association with cord blood cytokine concentrations.</a> Pediatr Res. 51, 310-316 (2002).</li><li>Jantzie, L. L., Corbett, C. J., Firl, D. J., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+Erythropoietin+Mitigates+Impaired+Cerebral+Cortical+Development+Following+Subplate+Loss+from+Prenatal+Hypoxia+Ischemia.">Postnatal Erythropoietin Mitigates Impaired Cerebral Cortical Development Following Subplate Loss from Prenatal Hypoxia Ischemia.</a> Cereb Cortex. , (2014).</li><li>Jantzie, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+attenuates+loss+of+potassium+chloride+co+transporters+following+prenatal+brain+injury.">Erythropoietin attenuates loss of potassium chloride co transporters following prenatal brain injury.</a> Mol Cell Neurosci. 61, 152-162 (2014).</li><li>Jantzie, L. L., Miller, R. H., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+signaling+promotes+oligodendrocyte+development+following+prenatal+systemic+hypoxic+ischemic+brain+injury.">Erythropoietin signaling promotes oligodendrocyte development following prenatal systemic hypoxic ischemic brain injury.</a> Pediatric Res. 74, 658-667 (2013).</li><li>Mazur, M., Miller, R. H., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+erythropoietin+treatment+mitigates+neural+cell+loss+after+systemic+prenatal+hypoxic+ischemic+injury.">Postnatal erythropoietin treatment mitigates neural cell loss after systemic prenatal hypoxic ischemic injury.</a> J Neurosurg Peds. 6, 206-221 (2010).</li><li>Robinson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+induced+by+graded+prenatal+systemic+hypoxic+ischemic+insults+in+rats.">Developmental changes induced by graded prenatal systemic hypoxic ischemic insults in rats.</a> Neurobiol Dis. 18, 568-581 (2005).</li><li>Delcour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroanatomical+sensorimotor+and+cognitive+deficits+in+adult+rats+with+white+matter+injury+following+prenatal+ischemia.">Neuroanatomical sensorimotor and cognitive deficits in adult rats with white matter injury following prenatal ischemia.</a> Brain Pathol. 22, 1-16 (2012).</li><li>Delcour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+prenatal+ischemia+on+behavior+cognitive+abilities+and+neuroanatomy+in+adult+rats+with+white+matter+damage.">Impact of prenatal ischemia on behavior cognitive abilities and neuroanatomy in adult rats with white matter damage.</a> Behav Brain Res. 232, 233-244 (2012).</li><li>Delcour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+musculoskeletal+and+locomotor+alterations+in+adult+rats+with+white+matter+injury+following+prenatal+ischemia.">Mild musculoskeletal and locomotor alterations in adult rats with white matter injury following prenatal ischemia.</a> Intl J Devel Neurosci. 29, 593-607 (2011).</li><li>Jantzie, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+pattern+of+interaction+between+in+utero+hypoxia+ischemia+and+intra+amniotic+inflammation+disrupts+brain+development+and+motor+function.">Complex pattern of interaction between in utero hypoxia ischemia and intra amniotic inflammation disrupts brain development and motor function.</a> J Neuroinflam. 11, 131 (2014).</li><li>Back, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+vulnerability+of+late+oligodendrocyte+progenitors+to+hypoxia+ischemia.">Selective vulnerability of late oligodendrocyte progenitors to hypoxia ischemia.</a> J Neurosci. 22, 455-463 (2002).</li><li>Jantzie, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+Expression+of+N+Methyl+d+Aspartate+(NMDA)+Receptor+Subunits+in+Human+White+and+Gray+Matter+Potential+Mechanism+of+Increased+Vulnerability+in+the+Immature+Brain.">Developmental Expression of N Methyl d Aspartate (NMDA) Receptor Subunits in Human White and Gray Matter Potential Mechanism of Increased Vulnerability in the Immature Brain.</a> Cereb Cortex. 25, 482-495 (2015).</li><li>Jantzie, L. L., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+Models+of+Encephalopathy+of+Prematurity.">Preclinical Models of Encephalopathy of Prematurity.</a> Devel Neurosci. , (2015).</li><li>Workman, A. D., Charvet, C. J., Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+transformations+of+neurodevelopmental+sequences+across+mammalian+species.">Modeling transformations of neurodevelopmental sequences across mammalian species.</a> J Neurosci. 33, 7368-7383 (2013).</li><li>Herlenius, E., Lagercrantz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+neurotransmitter+systems+during+critical+periods.">Development of neurotransmitter systems during critical periods.</a> Exper Neurol. 190, S8-S21 (2004).</li><li>Kelsom, C., Lu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+specification+of+GABAergic+cortical+interneurons.">Development and specification of GABAergic cortical interneurons.</a> Cell Biosci. 3, 19 (2013).</li><li>Kinney, H., Back, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+oligodendroglial+development+Relationship+to+periventricualr+leukomalacia.">Human oligodendroglial development Relationship to periventricualr leukomalacia.</a> Semin Pediatr Neuro. 5, 180-189 (1998).</li><li>Robinson, S., Li, Q., DeChant, A., Cohen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+amino+butyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma amino butyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg Peds. 104, 396-408 (2006).</li><li>Xu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+development+of+the+GABAergic+system+in+the+human+cerebral+cortex+and+white+matter.">Late development of the GABAergic system in the human cerebral cortex and white matter.</a> J Neuropathol Exp Neurol. 70, 841-858 (2011).</li><li>Segovia, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrested+oligodendrocyte+lineage+maturation+in+chronic+perinatal+white+matter+injury.">Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury.</a> Ann Neurol. 63, 520-530 (2008).</li><li>Robinson, S., Li, Q., Dechant, A., Cohen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+aminobutyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma aminobutyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg. 104, 396-408 (2006).</li><li>Boksa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prenatal+infection+on+brain+development+and+behavior+a+review+of+findings+from+animal+models.">Effects of prenatal infection on brain development and behavior a review of findings from animal models.</a> Brain Behav Immun. 24, 881-897 (2010).</li><li>Burd, I., Brown, A., Gonzalez, J. M., Chai, J., Elovitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+term+chorioamnionitis+unraveling+causes+of+adverse+neurological+outcomes.">A mouse model of term chorioamnionitis unraveling causes of adverse neurological outcomes.</a> Repro Sci. 18, 900-907 (2011).</li><li>Bergeron, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White+matter+injury+and+autistic+like+behavior+predominantly+affecting+male+rat+offspring+exposed+to+group+B+streptococcal+maternal+inflammation.">White matter injury and autistic like behavior predominantly affecting male rat offspring exposed to group B streptococcal maternal inflammation.</a> Devel Neurosci. 35, 504-515 (2013).</li><li>Uchida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ureaplasma+parvum+lipoprotein+multiple+banded+antigen+on+pregnancy+outcome+in+mice.">Effects of Ureaplasma parvum lipoprotein multiple banded antigen on pregnancy outcome in mice.</a> J Reprod Immunol. 100, 118-127 .</li></ol>

Os autores não têm nada a revelar.

Encefalopatia da prematuridade é difícil de modelar em animais por causa da complexa interação de etiologias, curso do tempo do desenvolvimento neurológico, complexidade da formação da rede cerebral humano, sobrepondo mecanismos de lesão, e os diversos fenótipos de CNS insulta manifesto em prematuros humanos. EoP está associada com as vulnerabilidades no tipo específico de célula (isto é, oligodendrócitos imaturos) 21, bem como diversas vias desenvolvente reguladas (isto é sub...

Com mais de 12% das crianças nascidas nos Estados Unidos antes da idade gestacional de 37 semanas estimada 1, lesão cerebral perinatal (PBI) da prematuridade é uma importante causa de incapacidade permanente. PBI da prematuridade, encefalopatia também denominado de prematuridade (EOP), afecta todo o sistema nervoso central (SNC). Lesão CNS muitas vezes começa no útero, e é agravada por processos de pré-natal, incluindo chorioamnionitis e complicações pós-natais, como hipóxia e sepse. PBI de insultos sistêmicos altera neurodesenvolvimento e leva à paralisia cerebral, epilepsia, atraso cognitivo e inúmeros transtornos neuropsiquiátricos afetando regulação emocional, memória e função executiva 1,2. Embora muito progresso tenha sido feito, uma compreensão limitada de como continua a ser as conseqüências celulares e moleculares da lesão CNS desde o nascimento prematuro traduzir para o grande número de sequelas neurológicas em crianças que nascem prematuros. Esta falta de conhecimento traseiraers diagnóstico em tempo real do CNS gravidade da lesão e dosagem informado das intervenções emergentes. Além disso, as estratégias terapêuticas adequadas à idade para essa população vulnerável paciente ainda imperceptíveis. Inflamação intra-uterino é muito comum na prematuridade extrema e envolve uma cascata inflamatória materno-fetal-placentária complexo 3. Infecção intra-uterina é frequentemente subclínica. Achados placentários específicos consistentes com inflamação aguda, ou corioamnionite histológica, são os principais determinantes da resposta inflamatória fetal e são coincidentes com lesão cerebral associada com o nascimento pré-termo 3-5. Na verdade, a resposta inflamatória fetal tem implicações clínicas distintas para resultados a longo prazo desde o nascimento prematuro. Os bebês que são pequenos para a idade gestacional (PIG) ​​ou que experimentam infecção são excepcionalmente vulneráveis ​​aos déficits neurológicos 3,4. Corioamnionite é um diagnóstico patológico típico após o nascimento prematuro <sup&gt; 6,7, e exame histológico revela sinais de inflamação em 70% das placentas de recém-nascidos muito prematuros 4. Além disso, corioamnionite é associado à disfunção cognitiva em dois anos 8. Evidência de subperfusão vascular materno na placenta de recém-nascidos prematuros extremos também está associada com paralisia cerebral na infância 9. O impacto sinérgico de corioamnionite e placentários defeitos de perfusão é bem ilustrado pelo notavelmente alto risco de resultados neurológicos anormais nessa população de pacientes aos dois anos de idade 10,11. Para imitar defeitos sistêmicos humanos placentária de perfusão e chorioamnionitis associadas com a inflamação induzida por patógeno, foi desenvolvido um modelo de pré-natal transitória sistêmica a hipóxia-isquemia (tshi) combinado com lipopolissacarídeo intra-amniótica (LPS) em ratos. Nosso objetivo foi adaptar o nosso modelo de tshi sozinho em ratos 12-16 para incluir a inflamação intra-uterina,para facilitar a modelagem pré-clínico de lesão do SNC associada com o nascimento prematuro. Tshi sozinho revelou perda persistente de células da linhagem oligodendrogliais, neurônios corticais, aumento da morte celular, e níveis elevados de citocinas pró-inflamatórias, com intervalos de isquemia progressivas que levam a um padrão de lesão classificada consistente com lesão cerebral pré-natal 16. Modificações isquémicos os componentes deste modelo também demonstraram défices de codificação da memória, a memória de curto e longo prazo e alterações músculo-esqueléticas leves em ratos com a idade 17-19. Na verdade, nós já demonstraram que a combinação de tshi + LPS recapitula as características fisiopatológicas da EoP, incluindo oligodendrócitos e perda neuronal, lesão axonal, inflamação celular e anormalidades funcionais 20.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Saline Solution, 0.9%</td>
			<td >Sigma</td>
			<td >S8776</td>
			<td ></td>
		</tr>
		<tr >
			<td >LPS 011B4</td>
			<td>Sigma</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr >
			<td >Evan&#39;s Blue Dye</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical gloves</td>
			<td>Biogel</td>
			<td>40870</td>
			<td></td>
		</tr>
		<tr >
			<td >OR Towels</td>
			<td>Cardinal Health</td>
			<td>287000-008</td>
			<td>Sterile</td>
		</tr>
		<tr >
			<td >PDI Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>06-669-62</td>
			<td></td>
		</tr>
		<tr >
			<td >Mini Arco Rechargeable Clippers</td>
			<td>Kent Scientific Corp.</td>
			<td>CL8787</td>
			<td></td>
		</tr>
		<tr >
			<td >Betadine surgical scrub</td>
			<td>Purdue Products L.P.</td>
			<td>67618-151-17</td>
			<td></td>
		</tr>
		<tr >
			<td >Eye Lubricant</td>
			<td>Refresh Lacri Lube</td>
			<td>00023-0312</td>
			<td></td>
		</tr>
		<tr >
			<td >Blunt Forceps</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td>Roboz</td>
			<td>RS-6808</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Scissors</td>
			<td>Roboz</td>
			<td>RS-5880</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Scissors</td>
			<td>F.S.T.</td>
			<td>14002-16</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td>1 ml</td>
		</tr>
		<tr >
			<td >Needle</td>
			<td>BD</td>
			<td>305122</td>
			<td>25G 5/8</td>
		</tr>
		<tr >
			<td >Needle</td>
			<td>BD</td>
			<td>305128</td>
			<td>30G 1</td>
		</tr>
		<tr >
			<td >Cotton-tipped Applicators</td>
			<td>Fisherbrand</td>
			<td>23-400-114</td>
			<td>Small, 6 inch sterile</td>
		</tr>
		<tr >
			<td >Cotton Gauze Sponge</td>
			<td>Fisherbrand</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr >
			<td >Needle Holders</td>
			<td>Kent Scientific Corp.</td>
			<td>INS600109</td>
			<td>12.5 CM STR</td>
		</tr>
		<tr >
			<td >Vessel Clips</td>
			<td>Kent Scientific Corp.</td>
			<td>INS600120</td>
			<td>30G Pressure</td>
		</tr>
		<tr >
			<td >3-0 Perma Hand Silk Sutures</td>
			<td>Ethicon</td>
			<td>1684G</td>
			<td>Black braided, 3-0 (2 metric), 18&#34;, non-absorbable,&#160; PS-1 24mm needle, 3/8 circle</td>
		</tr>
		<tr >
			<td >Insulin Syringes</td>
			<td>BD</td>
			<td>328438</td>
			<td>0.3cc 3mm 31G</td>
		</tr>
		<tr >
			<td >Pentobarbital</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Buprenorphine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Bupivacaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Lithium Carbonate</td>
			<td>Acros Chemicals</td>
			<td>554-13-2</td>
			<td></td>
		</tr>
		<tr >
			<td >Superfrost Plus Microscope Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr >
			<td >Hematoxylin</td>
			<td>Leica</td>
			<td>3801521</td>
			<td>Surgipath Gill II Hematoxylin</td>
		</tr>
		<tr >
			<td >Eosin</td>
			<td>Leica</td>
			<td>3801601</td>
			<td>Surgipath Eosin</td>
		</tr>
		<tr >
			<td >Xylenes</td>
			<td>Fisherbrand</td>
			<td>X3S-4</td>
			<td>Histological Grade</td>
		</tr>
		<tr >
			<td >Permount</td>
			<td>Fisherbrand</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr >
			<td >Coverglass</td>
			<td>Fisherbrand</td>
			<td>12-548-5P</td>
			<td>Fisher Finest Premium Coverglass</td>
		</tr>
	</tbody>
</table>

modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats

Encefalopatia da prematuridade (final do período) é um termo que engloba o sistema nervoso central (CNS) anormalidades associadas à prematuridade. Para melhores objetivos translacionais antecedência e descobrir novas estratégias terapêuticas para a lesão cerebral associada ao nascimento prematuro, modelos pré-clínicos de EoP deve incluir mecanismos semelhantes de lesões pré-natal mundial observada em humanos e envolvem múltiplos componentes do sistema materno-fetal-placentária. Idealmente, os modelos devem produzir um espectro semelhante de déficits funcionais no animal maduro e recapitular vários aspectos da fisiopatologia. Para imitar os defeitos humanos sistêmicas da placenta de perfusão, subperfusão placentária e / ou chorioamnionitis associadas com a inflamação induzida por patógeno em parto prematuro cedo, foi desenvolvido um modelo de pré-natal transitória sistêmica a hipóxia-isquemia (tshi) combinado com lipopolissacarídeo intra-amniótica (LPS). Em grávidas ratos Sprague Dawley, tshi via oclusão da artéria uterina on dia embrionário 18 (E18) induz um defeito subperfusão placentária graduada associada com o aumento da lesão do SNC do feto. Quando combinado com injeções de LPS intra-amniótica, inflamação da placenta é aumentada e danos CNS é agravado com a matéria, da marcha e de imagem brancos anormalidades associadas. Pré-natal tshi e tshi + LPS insultos pré-natal atender vários dos critérios de um modelo EoP incluindo recapitulando o insulto intra-uterina, causando perda de neurônios, oligodendrócitos e axônios, perda de subplate e déficits funcionais em animais adultos que imitam aqueles observados em crianças nascidas extremamente prematuro. Além disso, este modelo permite a dissecção da inflamação induzida por tipos de lesões divergentes.

Seguindo tshi + LPS em E18, hematoxilina e eosina revela anormalidades histopatológicas significativas, tanto na placenta (Figura 1) e no cérebro (Figura 2). Placentas examinadas em E19 e E21 são grosseiramente edematosa com micro-hemorragia e necrose em toda a decídua e labirinto. Também se observa infiltrado inflamatório significativo e aumento da vascularização. Cérebros examinados em P2 revelar ventriculomegalia, bem como a substância branca e perda subplate neurônio em c...

Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modelagem Encefalopatia da Prematuridade Usando pré-natal hipóxia-isquemia com Lipopolysaccharide intra-amniótica em Ratos

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

modeling-encephalopathy-prematurity-using-prenatal-hypoxia-ischemia

Research

JoVE Journal

Medicine

11.2K visualizações.  University of New Mexico. Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Vídeo: Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Curso de modelagem em Ratos - Oclusão da Artéria Cerebral Média com o modelo de Filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Medicina

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Imagem não invasivo e Análise de Isquemia Cerebral em Ratos de Vida Usando Positron Emission Tomography com<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Um Modelo de Leitão Neonatal encefalopatia hipóxico-isquêmica

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Avaliando Encefalopatia Espongiforme Transmissível Espécies Barreiras com uma<em&gt; In Vitro</em&gt; Prion Conversão Protein Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Simultânea de imagem PET / MRI Durante Rato isquemia-hipoxia cerebral

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Um Modelo de acidente vascular cerebral trombótico Baseado em Transient isquemia-hipoxia cerebral

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Circunscrito capsular infarto Modelagem usando uma técnica Photothrombotic

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Preparação de andaimes renais descelularizados em ratos

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Análise de Amostras de Cyperi Rhizoma Cru e Processadas Usando Cromatografia Líquida-Espectrometria de Massas em Tandem em Ratos com Dismenorreia Primária

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

Manipulação padronizada de Tuina para osteoartrite do joelho em ratos

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Autor em destaque: Compreendendo o mecanismo de manipulação da tuina em ratos 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Manipulação de Tuina para Reduzir a Inflamação e Perda de Cartilagem em Ratos com Osteoartrite de Joelho

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...