Name: a battery of motor tests in a neonatal mouse model of cerebral palsy
Uploaded: 2016-11-03T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy at JoVE.com

We would like to thank everyone at Shriners Hospital Pediatric Research Center, in particular Dr. Mickey Seltzer, of whom without his support, this work would not have been funded. In addition, we would like to thank Isha Srivastava, who contributed to early data collection and Amy He, who helped with the figures. This study was funded by Shriners Hospitals for Children. No funding source played a role in experimental design or decision to submit the paper for publication.

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The authors declare that they have no competing financial interests.

Usando modelos animais para estudar doenças humanas só é relevante se há uma sobreposição entre a resposta celular e molecular entre humanos e roedores e que os testes comportamentais realizados têm relevância direta para os sintomas humanos. Um dos principais problemas com doença pediátrica estudos é que muitos investigadores utilizar roedores adultos para criar o modelo, assim como a avaliação comportamental adulto roedor, sem ter em conta as diferenças no desenvolvimento que podem ser importantes para o processo da doença. Devido a estes problemas, é importante que a investigação sobre a utilização doença pediátrica, não só o desenvolvimento pontos de tempo apropriado ajustado (por exemplo, desenvolvimento do SNC humano com 28 - 32 semanas são equivalentes a um dia pós-natal 2-7 dias roedor) 7, mas também testes comportamentais que irá analisar motor apropriado, comportamentos de desenvolvimento sensoriais ou reflexivos. Assim, à medida que cada novo modelo de doença neonatal é desenvolvido, que devem ser rigorosamente testados para assegurar que o celulare respostas comportamentais irá fornecer os dados traduzíveis mais adequadas entre roedores e humanos. A paralisia cerebral é uma desordem motora, que persistem na vida adulta. Um problema com muitos dos modelos paralisia cerebral hoje disponíveis é a falta de repetibilidade, o teste de motor padronizado, que pode correlacionar-se com os défices observados em pacientes pediátricos. Neste novo modelo, que combina hipóxia, isquemia e inflamação em um rato neonatal, comportamento motor foi avaliada utilizando uma bateria de testes específicos para os ratos recém-nascidos. A fim de diminuir a subjectividade e aumentar a informação quantitativa, vários testes foram modificados para incluir muito específico, mas fáceis de avaliar as medidas que podem ser padronizados. Além disso, as avaliações front e dos membros posteriores podem ser feitas separadamente, e as diferenças para a esquerda / para a direita pode ser determinada. Esta bateria de testes é específico para ratos neonatais até duas semanas de idade. Este modelo demonstra CPdificuldade em andar (deambulação, ângulo do pé hindlimb), bem fraqueza específica do membro (suspensão de quatro membros, suspensão dos membros posteriores), e déficits de reflexos de desenvolvimento (agarrar reflex). Embora neste estudo apenas um ponto temporal foi examinada, esses déficits podem ser monitorados ao longo do tempo. Há outras baterias de testes que podem ser usados no recém-nascido, como a bateria do Fox de testes ou avaliação de marcos de desenvolvimento 15 de Heyser. No entanto, estes testes comparar o recém-nascido para o adulto, cujas respostas não podem ser o mesmo, porque o recém-nascido está ainda em desenvolvimento. A bateria do Fox e testes Assement de Heyser confiar na informação subjetiva observacional com (sim ou não) avaliação dicotômica, ao invés de dados objetivos (ângulo, postura com base na força, etc). Devido à subjetividade destes testes, muitos cientistas têm se adaptado, adicionados ou removidos critérios, tornando assim os seus resultados incomparáveis ​​para os outros e limiteing a utilidade dos dados em termos de estabelecimento de um défice de linha de base para uma determinada doença ou distúrbio. Ao estabelecer um conjunto de testes motores padronizados que são qualitativa e especificamente concebido para testar recém-nascidos, os resultados dos grupos de pesquisa individuais podem ser precisa e confiável relatados e comparados.

Desenvolvimento de novos modelos de lesão ou doença pediátrica usando roedores é muitas vezes difícil, devido à incrível capacidade de ambos os ratos e camundongos para se recuperar rapidamente da lesão neurológica. Portanto, a fim de validar qualquer novo modelo de doença pediátrica, examinando exaustivamente as alterações celulares e moleculares deve ir de mãos dadas com os resultados comportamentais. Em muitos aspectos, a recuperação comportamental funcional pode ser mais importante do que as alterações celulares subjacentes em termos de relevância terapêutica ou de translação. Enquanto os investigadores aprendem mais sobre lesão no adulto e neonatal, é claro que as suas respostas são muito diferentes e não podem ser extrapolados entre os dois. Por exemplo, ratinhos neonatais apresentar diferentes níveis de factor de crescimento de nervo, factor neurotrófico derivado do cérebro, neurotrofina-3 e factor neurotrófico glial da linha de células obtido de acordo com lesão da medula espinal 1,2. Além disso, os neonatos têm fugas barreira hemato-encefálica significativa após acidente vascular cerebral 3, demonstrate cortical neurônio rearranjo após a lesão do nervo periférico 4, e ter um astrogliosis atrasado ou diminuiu após lesão medular e hipóxia-isquemia 5,6. Portanto, é importante que a investigação pediátrica translacional usar modelos developmentally equivalente e que esses modelos são avaliados para ambas as alterações moleculares / celulares e testes comportamentais apropriadas à idade. Paralisia Cerebral (PC) é um distúrbio motor que afeta 3: 1000 nascidos vivos por ano (NIH). Crianças com PC exibem uma variedade de sintomas e condições co-mórbidas, dependendo da gravidade da doença. Dificuldade de movimento e coordenação são os sinais mais comuns, juntamente com atrasos para alcançar metas de desenvolvimento do motor. Outros sinais incluem o tônus ​​muscular anormal (aumentada ou diminuída), reduziu as habilidades motoras finas, dificuldade para caminhar, salivação excessiva e deglutição, e atrasos de fala (NIH). A causa subjacente da CP se acredita sera falta de oxigênio e / ou fluxo sanguíneo para o cérebro durante o período pré ou periparto, ou até um ano pós-parto. Além disso, a inflamação é agora acreditava ser um componente chave no desenvolvimento de CP. A maioria dos casos de PC estão associados com lesão da substância branca em torno dos ventrículos, conhecidos como leucomalácia periventricular (PVL). Esta característica neurológica sugere que o insulto inicial levando a CP ocorre durante o período de desenvolvimento do cérebro quando os oligodendrócitos são mais vulneráveis ​​ao insulto. O período de crescimento oligodendrócitos rápida em um ser humano, também o período em oligodendrócitos são os mais suscetíveis a lesões, é entre 24 - 32 semanas de gestação. No roedor, o período é equivalente dias pós-natais 2 - 7 7, e é quando PC é induzida neste modelo. O modelo de rato neonatal da CP que foi usado para realizar os testes descritos aqui combina hipoxia e isquemia com a inflamação de criar uma injUry que imita melhor a neurodegeneração visto em CP humano. Este modelo aborda algumas das principais deficiências observadas em outros modelos animais de CP, que não possuem déficits motores distintos que se assemelha a pacientes com PC humanos, bem como danos substância branca distinta. Estudos anteriores por um colaborador usando o mesmo modelo demonstraram que a adição de inflamação aumenta a lesão da substância branca, assim, emulando melhor o PVL visto em crianças com CP 8. Com base nos dados anteriores, este trabalho apresenta uma bateria completa de testes motores neonatais, a fim de avaliar as mudanças no comportamento do motor como o animal envelhece.

<table><tbody><tr><td></td><td></td><td></td><td></td></tr><tr><td>C57BL/6 mice</td><td>Charles River Laboratories</td><td>STRAIN CODE: 027&amp;nbsp;</td><td>C57BL/6NCrl is the exact strain we use</td></tr><tr><td>Anesthesia Dish, PYREX&amp;trade; Crystallizing Dish</td><td>Corning Life Sciences Glass&amp;nbsp;</td><td>3140125</td><td>Capacity: 25.03 oz. (740ml); Dia. x H: 4.92 x 2.55 in. (125 x 65mm). However, any small round glass container will work. A 2 cup capacity pyrex food storage bowl with flat bottom will also work and is much cheaper (Pyrex model number: 6017399).</td></tr><tr><td>Covered lead ring</td><td>Fisher Scientific</td><td>S90139C</td><td>Lead ring for stablizing flasks in a water bath. It is used inside the anesthesia dish.</td></tr><tr><td>Scalpel Blade #11</td><td>World Precision Instrucments, Inc.</td><td>500240</td><td></td></tr><tr><td>Small Vessel Cauterizer</td><td>Fine Science Tools</td><td>18000-00</td><td></td></tr><tr><td>Micro Hook</td><td>Fine Science Tools</td><td>10064-14</td><td></td></tr><tr><td>Vetbond Suture Glue</td><td>3M</td><td>1469SB</td><td>n-butyl cyanoacrylate adhesive</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma Life Science</td><td>L4391</td><td>Lipopolysaccaride from E.coli 0111:B4, gamma irradiated</td></tr><tr><td>12 x 12 inch opaque box</td><td>Acrylic Display Manufacturing: A division of Piasa Plastics</td><td>C4022</td><td>Colored Acrylic 5-Sided Cube, 3/16&quot; Colored Acrylic, 12&quot;W x 12&quot;D x 12&quot;H;&amp;nbsp; http://www.acrylicdisplaymfg.com/html/cubes_19.html</td></tr><tr><td>Camera/camcorder</td><td>JVC</td><td>GC-PX100BUS</td><td>Any camcorder that works well in low light and can be imported and edited. We use the JVC GC-PX100 Full HD Everio Camcorder.</td></tr><tr><td>Covidien Tendersorb&amp;trade; Underpads</td><td>Kendall Healthcare Products Co</td><td>7174</td><td></td></tr><tr><td>WypAll L40</td><td>Kimberly-Clark Professional</td><td>5600</td><td>Any surface with moderate grip will do</td></tr><tr><td>Surface at 45 degree incline</td><td></td><td></td><td>We use a cardboard box.</td></tr><tr><td>Thin wire from a pipe cleaner</td><td>Creatology</td><td>M10314420</td><td>Any pipe cleaner from any craft store will work.</td></tr><tr><td>50mL conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Fiberglass Screen Wire</td><td>New York Wire&amp;nbsp; www.lowes.com</td><td>14436</td><td>Any supplier can be used as long as their screen is 16 x 16 or 18 x 16</td></tr><tr><td>Razor blade</td><td>Fisherbrand</td><td>12-640</td><td>A wooden stick applicator or wooden part of a cotton-tipped swab will also work.</td></tr><tr><td>OPTIX 24-in x 4-ft x 0.22-in Clear Acrylic Sheet to make Clear Acrylic Walkway</td><td>PLASKOLITE INC</td><td>1AG2196A</td><td>Clear acrylic (1/8&quot; thick) with sides and a top to limit exploration. We bought a sheet of acrylic from a local hardware store and had them cut it to size. (2) 2&quot; x 2&quot;; (3) 2&quot; x 18&quot;; (1) 2&quot; x 15.5&quot;; (1) 2&quot; x 3&quot;. Using clear tape, tape all sides together, with the 15.5&quot; piece on top. Tape the 3&quot; piece to the end of the 15.5&quot; piece to create a flap/entryway for the mice. Alternatively, part or all of the walkway can be glued together, and only taping on the top pieces. This design will allow for the walkway to be opened for easy cleaning.</td></tr><tr><td>Protractor</td><td>Westscott</td><td>ACM14371</td><td></td></tr></tbody></table>

a battery of motor tests in a neonatal mouse model of cerebral palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Os ratos foram testados a partir P7 (24 h após a cirurgia) para P13 (1 semana após a cirurgia), utilizando camundongos diferente para cada ponto de tempo para que a aprendizagem de uma paradigma de teste não foi uma variável de confusão. P8 foi seleccionado como resultados representativos, como os ratos apresentaram as maiores défices neste ponto de tempo. Transição de rastejar para caminhar está atrasado em CP Mice Neonatal pacientes com PC humanos têm anormalidades da marcha, que vão desde toe-pé para uma marcha scissored. Como este CP modelo exibe marcha déficits semelhantes aos seres humanos, deambulação foi avaliada. Os ratos foram marcados na simetria da marcha e os movimentos dos membros-pata durante uma caminhada em linha reta. Em 48 horas após a cirurgia (PND 8), os ratos CP tinha menos movimento do membro simétrica e um &quot;rastreamento&quot; marcha em comparação com os seus homólogos sham (pontuação média deambulação: CP 1.083 ±0,6337, n = 12 vs sham 1.639 ± 0,4859, n = 9; p &lt;0,05, Figura 10). Por uma semana, ambos CP e ratos sham fez a transição para uma curta (dados não mostrados). <img alt="Figura 10" src="/files/ftp_upload/53569/53569fig10.jpg" /> Figura 10. CP mouse filhotes de cachorro não deambular, bem como Shams. Ratos Sham (barra preta) têm uma pontuação média de 1.639 ± 0,4859 (n = 9), ou seja, seu desenvolvimento ambulatorial cai entre os movimentos dos membros assimétrica e rastreamento lento. camundongos CP (barra cinza) recebem uma pontuação média de 1.083 ± 0,6337 (n = 12), ou seja, a sua deambulação é menos desenvolvida e tendem a ter os movimentos dos membros assimétrica. Os dados são expressos como média ± SEM; * É p &lt;0,05. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53569/53569fig10large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Hindlimb Pé Ângulo é aumentada em CP Além de deambulação, hindlimb pé-angular foi avaliada. Oito dias de idade filhotes de rato sham caminhar com suas patas traseiras virada para a frente, em comparação com camundongos LIS, que têm espalhados patas traseiras ao andar em uma linha reta (Figura 2; ângulo médio: CP 77,48 ± 9,848, n = 9, vs sham 54,54 ± 8,043, n = 11; p &lt;0,0001, Figura 11). Este aumento correlaciona-se com ângulo instabilidade da marcha, em que os filhotes necessidade de aumentar o ângulo de suas patas traseiras, a fim de estabilizar a sua marcha e auxiliar com o equilíbrio e coordenação. <img alt="Figura 11" src="/files/ftp_upload/53569/53569fig11.jpg" /> Figura 11. CP mouse filhotes de cachorro Splay sua patas traseiras ao andar. Camundongos CP (barras pretas) têm um ângulo médio entre os seus membros posteriores de 77,48 ± 8,043 (n = 11), enquanto que os ratos sham (cinza b<html> ars) têm um ângulo médio de 54,54 ± 9,848 (n = 9). Os dados são expressos como média ± SEM; **** É p &lt;0,0001. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53569/53569fig11large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Ratos CP não apresentar déficits quando Righting Superfície O teste de endireitamento superfície foi incluído como alguns pacientes com PC têm prejudicado controle de tronco (Heyrman et al., 2013). Além disso, o sistema vestibular é necessário para detectar a necessidade de corrigir e há déficits vestibulares em alguns pacientes com PC 23. ratinhos CP não apresentam défices significativos quando endireitamento, em comparação com controlos falsos (dados não mostrados). Ratos CP executam a mesma que Sham em geotaxis Testing Negative t &quot;fo: manter-together.within-page =&quot; 1 &quot;&gt; geotaxis negativo é usado para testar a coordenação motora em jovens filhotes dos ratos são desafiados por ser lugar de frente para baixo em uma superfície inclinada atraso ou falha na subida oriente poderia indicar déficits.. em coordenação, equilíbrio, ou entrada vestibular. camundongos CP não apresentam déficits quando desafiado com geotaxis negativos em comparação com os ratos sham (dados não mostrados). Além disso, os ratos CP não mostram uma preferência para virar para um lado contra o outro, quando re-orientação . Front-Membro teste de suspensão é apropriado para ratos mais velhos do que 10 dias pacientes com PC têm diminuição do tônus ​​muscular e déficits de habilidades motoras finas, como agarrar. Para testar a fraqueza neste modelo de ratinho, foi utilizado um teste de suspensão front-membro. Além disso, este modelo usa lesão isquêmica unilateral e lados-ness poderia ser determinada usando este teste suspensão. Esse testeé melhor para camundongos com idade superior a 10 dias 15. Aos 8 dias de idade, dois dias após a lesão, não existiram diferenças significativas entre CP e ratos sham (dados não mostrados). Força hindlimb é diminuída em camundongos CP pacientes com PC humanos muitas vezes precisam de chaves ou dispositivos de assistência de passeio devido à falta de controle motor e força. A fim de comparar o modelo CP roedor para os seres humanos, a força dos membros posteriores foi avaliada utilizando o teste da suspensão dos membros posteriores. Quando suspenso a partir do lado de um tubo cônico, ratos CP mostrou fraqueza dos membros posteriores, como demonstrado por uma diminuição na suspensão pontuação (hindlimb pendurado pontuação: CP 3,468 ± 0,5561, n = 13, vs sham 3,891 ± 0,1329, n = 13; p &lt; 0,05, Figura 12). Não foi observada diferença no tempo de suspensão dos membros posteriores (dados não mostrados). Assim, semelhante a pacientes com PC humanos, ratos CP demonstrar hindlIMB (perna) fraqueza. <img alt="Figura 12" src="/files/ftp_upload/53569/53569fig12.jpg" /> Figura 12. Os ratos Sham são ligeiramente mas significativamente mais forte em seus membros posteriores do que os ratos CP. Em uma pontuação média de suspensão de 3,891 ± 0,1329 (n = 13), os ratos sham (barra preta) mostram separação mais hindlimb, e, portanto, uma postura hindlimb mais forte, quando pendurado na borda de um tubo do que os ratos CP (barra cinza) com uma pontuação média de suspensão de 3,468 ± 0,5561 (n = 13). Os dados são expressos como média ± SEM; * É p &lt;0,05. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53569/53569fig12large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Força de preensão é diminuída após lesão CP Agarrando com todas as quatro patas é importante para um roedortermos de escalada e corrida em superfícies irregulares. Aderência exige força sustentada significativa, ao invés de destreza ou força linear, principalmente nos dígitos e as patas 24. Os ratos foram obrigados a manter seu peso corporal em uma tela de malha de arame invertido. Camundongos CP não puderam manter a sua aderência e estes ratinhos caiu em ângulos significativamente mais baixos (quatro membros ângulo média: CP 75,627 ± 24,48, n = 11, vs sham 96,57 ± 10.836, n = 9; p &lt;0,05, Figura 13). Estes dados mostram que há um défice significativo na força de preensão em ratinhos CP. <img alt="Figura 13" src="/files/ftp_upload/53569/53569fig13.jpg" /> Figura 13. Ratos CP têm aperto mais fraco do que Shams. Ratos Sham (barra preta) pode agarrar a um ângulo invertido média de 96,57 ± 10.836 (n = 9). camundongos CP (barra cinza) só pode chegar a um ângulo invertido de 75,627 ± 24,48 (n = 11). Os dados são expressos como média ± SEM; * P é0; 0.05. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53569/53569fig13large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Déficits apreensão Reflex são aparentes em Ratos CP Junto com deficiências motoras graves, movimentos motoras finas também são prejudicadas em pacientes com PC 25,26. O reflexo de agarrar em seres humanos está presente ao nascimento e desaparece em torno de 5-6 meses. No entanto, as mudanças no reflexo agarrar, como a velocidade exagerada ou a força de agarramento, incapacidade de compreender, ou o ressurgimento do reflexo de agarrar depois de 6 meses de idade, todos indicam danos ao sistema nervoso. Para comparar agarrar no modelo CP, foram determinados déficits agarramento reflexivos. Em 48 horas após a lesão, os ratos CP demonstrar uma diminuição na agarrando reflex (patas média apreendidoàs 48 horas: CP 2,429 ± 0,9376, n = 14, vs sham 3.214 ± 0,8018, n = 14; p &lt;0,05, Figura 14A). Houve um aumento ligeiro, mas não significativo, de preferência pata direita nas patas dianteiras (dados não mostrados). Houve uma preferência pata direita significativa nas patas traseiras (CP 75,0 ± 42,74, n = 14, vs sham 17,86 ± 54,09, n = 14; p &lt;0,005, Figura 14B). Uma semana após a lesão, os ratos CP apresentar déficits agarrando (patas média apreendido em 1 semana: CP 2,75 ± 1,035, n = 8, vs sham 3,80 ± 0,6325, n = 10; p &lt;0,05, Figura 14C), com nenhuma preferência pata notável . <img alt="Figura 14" src="/files/ftp_upload/53569/53569fig14.jpg" /> Figura 14. Ratos CP têm déficits agarrar, no patas traseiras, contralateral à região do cérebro ferido. (A) 48 horas após a lesão (PND 8), os ratos CP (barra cinza) compreender uma vara com, em média, menos patas do que anima shamls (barra preta). ratinhos (B) CP (barra cinza) exibem uma preferência para agarrar com a pata traseira direita (contralateral à lesão) em vez de usar a pata traseira esquerda (ipsilateral à lesão). ratos Sham (barra preta) não apresentam essa preferência pata direita. Preferência pata direita é calculado como ([pata direita - esquerda pata] / [pata direita + pata esquerda + ambas as patas] * 100) (C) Uma semana após a lesão, os ratos CP (barra cinza) mostram ainda agarrando déficits em comparação com. shams (barra preta). Os dados são expressos como média ± SEM; * É p &lt;0,05, ** é p &lt;0,005. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53569/53569fig14large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Ratos CP se afastar da borda Durante Aversão Cliff O teste baseia-se na aversão rochedo o medo inerente dos ratinhos para ligar AWAy de um penhasco íngreme e cabeça para a segurança. Embora alguns pacientes com PC têm dificuldades vestibulares, bem como o controle motor prejudicada, os ratos CP não apresentaram déficits neste teste.

Watch this Scientific Journal Video about A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy at JoVE.com

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

Uma bateria de testes de motor em um Neonatal Rato Modelo de Paralisia Cerebral

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

a-battery-of-motor-tests-in-a-neonatal-mouse-model-of-cerebral-palsy

Universidad San Sebastian

Research

JoVE Journal

Medicine

24.0K visualizações.  Lewiz Katz School of Medicine at Temple University. Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

Vídeo: A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Avaliação da função sensório-motora em rato modelos de doença de Parkinson

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Comportamento

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed &#8220;paw-dragging&#8221;. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia &#8211; two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

Pata-Arrastando: a Novel, análise de sensibilidade do mouse cilindro de ensaio

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Medindo Coordenação Motora em Ratos

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Neurociência

Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are also referred to as primitive reflexes. They are sensitive and consistent with later outcomes. Abnormal reflexes are described as an absence, persistence, reappearance, or latency of reflexes, which are predictive indices of infants that are at high risk for neurodevelopmental disorders. Animal models of neurodevelopmental disabilities, such as cerebral palsy, often display aberrant developmental reflexes, as would be observed in human infants. The techniques described assess a variety of neurodevelopmental reflexes in neonatal rats. Neurodevelopmental reflex testing offers the investigator a testing method that is not otherwise available in such young animals. The methodology presented here aims to assist investigators in examining developmental milestones in neonatal rats as a method of detecting early-onset brain injury and/or determining the effectiveness of therapeutic interventions. The methodology presented here aims to provide a general guideline for investigators.

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

Desenvolvimento neurológico Reflex Teste em Neonatais rato filhotes de cachorro

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are ...

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

Bateria de comportamental testes de avaliação geral de locomoção, coordenação e força Muscular em camundongos

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

A Neonatal do rato Spinal Cord Injury Compression Modelo

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

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 Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Um modelo de Mouse controlado para sepse Neonatal Polymicrobial

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Imunologia e Infecção

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Um modelo de mouse de lesões cerebrais traumáticas leves simples e repetitivas

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

Quantificando a disfunção locomotora em camundongos com lesão medular usando o MouseWalker

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

Autor em destaque: Usando o MouseWalker para quantificar a disfunção locomotora em um modelo de camundongo com lesão medular  

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.