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>

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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>

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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.

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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

Universidad de Cartagena UDEC

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

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

Modelos de mouse de leucomalácia periventricular

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Neurociência

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

O Modelo de encefalopatia hipóxico isquêmica Perinatal da Isquemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biologia

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 ...

Medicina

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Neurocomportamentais avaliações em um modelo do rato de lesão cerebral hipóxica-isquêmica Neonatal

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Um modelo do rato de leve hipoperfusão intra-uterino com estenose Microcoil

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

Um modelo de furão de inflamação sensibilizada tardia preterm-isquêmica lesão cerebral

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

Modelagem da Hemorragia Intraventricular Neonatal Através da Injeção Intraventricular de Hemoglobina

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Modeling Posthemorrhagic Hydrocephalus of Prematurity in Rats

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 weeks gestational age (GA). PHHP is defined by the accumulation of cerebrospinal fluid (CSF) associated with clinical symptoms of elevated intracranial pressure (ICP). Infants with PHHP suffer lifelong shunt dependence, with half requiring repeat surgery in the first year of life and many requiring multiple additional surgeries throughout the lifespan. Prenatal chorioamnionitis predisposes preterm infants to severe IVH and the need for surgical treatment of PHHP trends with neonatal sepsis. These clinical features suggest that systemic inflammation is an integral component of PHHP pathophysiology. 
Here, we define an animal model that recapitulates all clinical aspects and essential features of PHHP in rats. The goal of this protocol is to illustrate how in utero chorioamnionitis and postnatal IVH using lysed red blood cells can be combined to yield PHHP. This preclinical approach yields progressive macrocephaly and domed craniums, elevated intracranial pressure, and ventriculomegaly that can be detected via magnetic resonance imaging (MRI) or via microscopy. In addition to sustained disruption in CSF dynamics, rats also have cognitive delay and functional disability into adulthood. Accordingly, this preclinical platform facilitates unique and unparalleled translational studies of PHHP that can incorporate molecular, cellular, biochemical, histologic, imaging, and functional outcome measures. It can also be used for rigorous analysis of the choroid plexus, ependymal motile cilia, and glymphatic system in parallel. Last, it can also be an invaluable preclinical tool for the investigation of novel surgical intervention strategies and non-surgical therapeutic approaches for the treatment of hydrocephalus.

Posthemorrhagic hydrocephalus of prematurity (PHHP) can be modeled in neonatal rats by combining chorioamnionitis and intraventricular hemorrhage. The combination of these prenatal and postnatal events accurately recapitulates the clinical hallmarks of PHHP, including macrocephaly, ventriculomegaly, and elevated intracranial pressure, through the lifespan.

Modelagem da hidrocefalia pós-hemorrágica da prematuridade em ratos

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 ...

Induction of Cerebral Microhemorrhages in a Rat Model Using Lipopolysaccharide

Source: Li, D., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58423">Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing blood-brain barrier (BBB) disruption and cerebral microhemorrhages in a rat model using lipopolysaccharide (LPS). It highlights the sequence of immune responses that lead to increased BBB permeability and subsequent brain tissue damage.

Indução de micro-hemorragias cerebrais em modelo de rato usando lipopolissacarídeo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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