Este trabalho foi apoiado pelo National Institutes of Health [Grant não. R01NS069726] e da Associação Americana do Coração, em Grant Grant Aid [Grant não. 13GRNT17020004] para SD.

Nota: Os ratinhos (C57BL / 6J, macho) envelhecido 10 - 12 semanas, foram utilizados neste estudo. Todos os procedimentos foram realizados de acordo com o Guia NIH para o Cuidado e Uso de Animais de Laboratório e foram aprovados pela Universidade de Missouri Animal Care Institucional e Comitê de Uso (IACUC). 1. Pré-Surgery <ol><li> No dia antes da cirurgia autoclave e esterilizar todos os instrumentos cirúrgicos. Enrole os instrumentos e autoclave a 121 ° C a 15 psi durante 30 min seguido por 30 min de secagem (121 ° C, 15 psi, 30/30 ciclo). Coloque os instrumentos em um ambiente limpo e esterilizado até à sua utilização. </li><li> Solução fresca Rosa Bengala (RB) (20 mg / ml em solução salina estéril) toda vez que antes da cirurgia se preparar. Para dissolver completamente RB, o tubo de vórtice, em seguida, seguido por sonicação durante 5 min a 50/60 Hz, com potência de 19 W. Envoltório do tubo em folha de alumínio e proteger da luz de saída até novo utilizado durante surGery. </li><li> Prepara-se uma mistura de cetamina / xilazina em solução salina estéril. Adicionar 125 ul de xilazina (concentração estoque: 20 mg / ml) e 325 ul de ketmaine (concentração estoque: 100 mg / ml) e 550 ul de solução salina estéril para perfazer um volume final de 1 mL de mistura de anestésicos. </li><li> Pré-aquecer a almofada de aquecimento homeotérmicos. </li><li> Pré-aquecer a lâmpada de iodetos metálicos (fonte de luz para FN1 epi-microscópio de fluorescência) durante 30 minutos para estabilizar a potência da lâmpada. </li><li> Ajustar o tamanho da região iluminada a um diâmetro de 1 mm, utilizando uma objectiva 10X e retículo ajustando o diafragma de campo na vertical FN1 epi-microscópio de fluorescência. </li></ol> 2. Procedimento Cirúrgico <ol><li> Anestesiar o rato com uma dose de cetamina (130 mg / kg B. peso.) E xilazina (10 mg / kg B. peso.). Com base no cocktail em 1,3, 4 ul por g de rato B. peso. são precisos. Esterilizar o local da injecção com um algodão com álcool e administrar os anestésicos através do intra-peritoneal rota (IP). Tome cuidado para não injetar os anestésicos para os vasos sanguíneos do músculo ou como isso iria atrasar a indução e recuperação do animal. </li><li> Aplicar pomada lágrima artificial para ambos os olhos do rato para evitar a secagem e colocar o animal no almofada de aquecimento para evitar a hipotermia. </li><li> Preparação de animais <ol><li> Verifique o animal para a anestesia adequada nível cirúrgico usando a resposta toe pitada. </li><li> Uma vez que o animal atingiu o nível cirúrgico de anestesia, cortar o cabelo na superfície dorsal em torno da linha média do animal usando um aparador de cabelo elétrico. Esfregar o local cirúrgico com etanol a 70% seguido por solução de betadine três vezes. Cubra o local com um campo cirúrgico estéril até o próximo passo. </li></ol></li><li> Procedimento cirúrgico para afinar o osso para expor a medula espinhal <ol><li> Colocar o rato em uma posição de bruços através da almofada de aquecimento homeotérmica na plataforma cirúrgica (Figura 1A). Corretamente proteger o ratopostura usando uma braçadeira de focinho para manter uma região de pescoço alongado (Figura 1A, B). </li><li> Faça uma incisão (cerca de 1 cm de comprimento) usando tesouras cirúrgicas ao longo de meados da linha dorsal que se estende desde o T9 vértebras torácicas para T12. Afastar a pele para expor a área cirúrgica. </li><li> Usando um bisturi, cuidadosamente limpar o músculo para expor as espinhas dorsais na T9 - T12 vértebras. Parar o sangramento em cada etapa aplicando uma pressão suave com cotonete estéril. T10 separado - T12 vértebras do músculo circunvizinho e fixá-los usando uma braçadeira vertebral para estabilizar e impedir qualquer movimento (Figura 1A, B). </li><li> Usando uma broca de alta velocidade com osso polimento broca, com cuidado e fina suavemente a superfície dorsal da vértebra T10 ou T11 para visualizar a veia vertebral posterior e outras embarcações pequenas na superfície dorsal do SC (Figura 1C). </li><li> Para evitar danos térmicos, devido ao calor gerado durante o processo de desbaste,aplicar uma corrente suave e constante de solução salina normal juntamente com sucção constante para remover os detritos. </li><li> Usando um bisturi cuidadosamente alisar a superfície do osso até que o recipiente principal é claramente visível. Tome cuidado para não danificar a medula espinhal durante este processo. </li><li> Uma vez que o vaso sanguíneo é visualizado, RB administrar a uma dose de 30 mg / kg (peso corporal) por via do seio retro-orbital usando uma seringa de insulina. </li><li> Medir o fluxo sanguíneo através de um medidor de fluxo laser de Doppler após 3 min após a injecção, se necessário, RB (Figura 2A, B). Manter a assepsia durante o procedimento inteiro. </li></ol></li></ol> 3. Indução de PT <ol><li> Colocar o animal numa fase ajustável posição XY ao longo de um Lab-Jack que pode ajustar a altura. Ajuste a posição do mouse para que a região exposta do T11 medula espinhal está diretamente sob a objetiva de 10x do microscópio FN1 epi-fluorescência (Figura 3A). </li><li> Defina o poder do lifonte GHT a 12% e irradiar a região T11 com um diâmetro de 0,75 milímetros no meio da medula espinal diluído (Nota: esta região inclui a veia vertebral posterior e outros capilares) com uma luz verde (comprimento de onda de 540-580 nm, que é conseguido pelo cubo de filtro no microscópio) através da objectiva 10X durante 2 min. Captar imagens no início e no fim de irradiação (Figura 3B, C) ​​e registar o tempo da experiência neste ponto. </li><li> Medir o fluxo de sangue de novo durante 10 minutos, se necessário, através da colocação da sonda Doppler de laser para a mesma posição acima da medula espinhal como em 2.4.8 (Figura 2A, B). </li><li> Após o check-irradiação para qualquer hemorragia e se nenhum encontrado proceder à sutura do animal. Sutura da fáscia superficial juntamente com os músculos de cada lado da medula espinhal do usando uma sutura absorvível ou tamanho 4-0 sutura de seda. Tome cuidado para não danificar o SC exposta. Sutura da pele com fio de seda 4-0. Aplicar Betadine ou iodopara as bordas da pele após a sutura. </li></ol> 4. Pós-cirurgia Cuidados <ol><li> Após a sutura, coloque o animal na almofada de aquecimento para a recuperação. Após a recuperação dos animais para verificar sinais de déficits neurológicos, observando o movimento de ambas as traseiras-membros. Não deixe o animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. </li><li> Transferir os animais para a gaiola. Não devolva o animal que foi submetido a cirurgia para a companhia de outros animais até que esteja totalmente recuperado. </li><li> Confira os animais em intervalos regulares. Em caso de déficits neurológicos graves, fornecer o cuidado adequado, como evacuação de bexiga, administração de analgésicos (buprenorfina, 0,05-0,1 mg / kg). Verifique se há desidratação e administrar por via subcutânea soro fisiológico normal em caso grave. Normalmente, a buprenorfina (0,1 mg / kg), será administrada após a sutura para aliviar a dor no local da cirurgia. </li><li> Se os animais não são imediatamentesacrificados após a cirurgia, vamos colocar alto teor de água dieta no chão da gaiola, para que os animais possam alcançar o alimento facilmente. </li></ol> 5. transcardial Perfusão, coloração Nissl e Immunostaining <ol><li> Transcardialmente perfundir o animal, como descrito anteriormente 17-20. <ol><li> Anestesiar o animal, tal como descrito anteriormente no protocolo e perfundir transcardíaca com solução salina tamponada com fosfato (PBS, pH 7,4), seguido de gelado paraformaldeído a 4% (PFA) em PBS. </li><li> Após a perfusão, remover a medula espinhal (SC) e pós fixar-lo em PFA 4% em PBS a 4 ° CO / N. Transferir o SC fixo em PBS com 30% de sacarose e mantê-lo por 2 - 3 dias até que se deposita no fundo do tubo. </li><li> Usando um criostato corte da espinal medula em 30 mm de espessura e colocá-los em série em um revestido com gelatina em lâminas de vidro ou uma placa de 48 poços com 0,01 M de PBS. </li></ol></li><li> Coloração de Nissl: Para inspecionar o prejuízo causado pela PT realizar uma Nissl staining em secções da espinal medula como descrito previamente 17-20. <ol><li> Resumidamente, recolher cada quinta fatia da medula espinhal nas lâminas de vidro e mancha com 0,25% de cresilo violeta. Tome imagens dos cortes corados (Figura 4). </li></ol></li><li> A imunocoloração: Tal como anteriormente descrito utilizando um método secção 17,18,20 flutuante. <ol><li> Resumidamente, corar as secções da espinal medula por incubação O / N a 4 ° C com anticorpo de coelho anti-glia proteína fibrilar ácida (GFAP) anticorpo policlonal (1: 300), anticorpo de coelho anti-NeuN (1: 300), e anti-coelho anticorpo Iba1 (1: 500) seguido de anti-coelho de burro conjugado com Alexa 568-IgG (1: 400) de anticorpos secundários durante 4 h à TA. Captar imagens com um microscópio de fluorescência (Figura 5). </li></ol></li></ol>

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The authors have nothing to disclose.

Neste estudo, descrevemos um modelo photothrombotic de SC isquemia. Devido aos avanços na engenharia genética, tem havido uma onda de camundongos transgênicos comercialmente disponível que tornou possível estudar o impacto de genes específicos envolvidos na fisiopatologia isquêmico no SC. O objectivo do estudo foi o de desenvolver um modelo reprodutível rato de isquemia medular. Aqui nós adaptamos um modelo cortical PT para induzir SCI em camundongos. No seguimento da cirurgia da coluna vertebr...

Traumática lesão medular (LM) é uma condição clínica devastador que afeta as funções sensório-motoras e autonômicas do SC. Pacientes sobreviventes SCI muitas vezes são deixados com paraplegia debilitante que afeta significativamente suas atividades diárias e qualidade de vida 1. Modelos Experimental SCI têm sido uma ferramenta indispensável na investigação científica para compreender a fisiopatologia da SCI e processos de reparação neural associados. Estes modelos também foram utilizados para testar a eficácia pré-clínica de diversas intervenções neuroprotectores experimentais que são destinadas a recuperação funcional. Atualmente, a maioria dos modelos SCI, na prática, empregar o uso da força física sem corte para perturbar e prejudicar o SC mecanicamente. Estes métodos incluem contusão, compressão, deslocamento e transecção do SC 2. Tem sido sugerido que após o insulto mecânica primária uma lesão secundária sob a forma de conjuntos de isquemia no lesada no SC<html> 3,4. A etiologia da isquemia secundária inclui extensa degeneração do tecido, hemorragia parenquimatosa e às vezes por obstrução de vasos sanguíneos por edema do tecido 5-7. Como resultado da lesão secundária a integridade de SC é mais afectada, neurónios e células gliais são severamente prejudicada em função e viabilidade e sofrem apoptose que leva a um crescimento de infarto durante a fase crónica da lesão, análogo ao crescimento de penumbra de isquemia pós-AVC 8,9. Vários mecanismos como excitotoxicidade, produção de radicais livres, e inflamação foram relatados para ser responsável pela morte celular isquémica após uma lesão medular 10,11. Além disso, SC isquemia é uma complicação grave de cirurgias tóraco-abdominal do aneurisma da aorta, que muitas vezes levam a paraplegia nos pacientes 12,13. Apesar de tão alto impacto clínico muito poucos modelos de isquemia medular com alta reprodutibilidade estão disponíveis no momento. nt &quot;&gt; Indocianina (PT) é um método vulgarmente utilizado para a indução de isquemia focal no cérebro 14-20. A técnica é relativamente não invasivo e altamente reprodutível e produz uma lesão isquémica focal exacta da área exposta do cérebro 17 -21. Isto é conseguido por administração sistémica de corantes fotoactivos como Rose Bengal (RB) ou 16-20,22 eritrosina B 23 seguido por irradiação localizada de vasos sanguíneos com fonte de luz adequada. A fotoactivação do corante faz com que a geração de radicais livres que interromper a integridade do endotélio vascular liso, e fazer com que as plaquetas se acumulam, que subsequentemente forma um trombo. A obstrução do fluxo de sangue pelos resultados do trombo em um enfarte na região fornecido pelo vaso 24. Devido à facilidade de controlo sobre o intensidade e da duração da irradiação este procedimento produz um enfarte altamente uniforme e reprodutível. Além disso, este método pode ser empregue para induzir um enfartet em várias posições anatómicas permitindo espacial (por exemplo, substância cinzenta vs matéria branca) compreensão do efeito da isquemia. O objetivo do presente estudo é desenvolver um modelo fácil e altamente reprodutível de SC isquemia em ratinhos. Nós descrevemos o procedimento de um modelo PT de SC isquemia em ratinhos. Os resultados de histologia e imunocoloração demonstrou que a PT pode efectivamente induzir enfarte do SC, perda neuronal e gliose reactiva.

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			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
			<td style="width:233px;">Sigma-Aldrich</td>
			<td style="width:180px;">330000</td>
			<td style="width:421px;">20 mg/ml in sterile saline</td>
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			<td height="21" style="height:21px;">C57Bl6/J</td>
			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
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			<td height="21" style="height:21px;">Ketamine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-996-06</td>
			<td>100 mg/ml</td>
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			<td height="21" style="height:21px;">Xylazine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-234-11</td>
			<td>100 mg/ml</td>
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			<td height="21" style="height:21px;">Betadine solution</td>
			<td>Purdue</td>
			<td>NDC-67618-150-01</td>
			<td>10% povidone iodine topical solution</td>
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			<td height="21" style="height:21px;">Normal saline</td>
			<td>Abott Laboratories</td>
			<td>04930-04-10</td>
			<td>For diluting RB, anaesthesia and for preventing tissue from drying</td>
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			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
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			<td>EXFO, Canada</td>
			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
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			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td>Fine Science Tool</td>
			<td>15000-10</td>
			<td>for minor dissection</td>
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			<td>Fine Science Tool</td>
			<td>14063-011</td>
			<td>No. 3 handle</td>
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			<td>Fine Science Tool</td>
			<td>10003-12</td>
			<td>for removing muscle</td>
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			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
			<td>2976</td>
			<td>No. 10</td>
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			<td height="21" style="height:21px;">Forceps (curved)</td>
			<td>Fine Science Tool</td>
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			<td height="21" style="height:21px;">Tissue adhesive glue</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>to adhere to edges of the cut skin</td>
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			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
			<td>USSC Sutures</td>
			<td>VP-521</td>
			<td>Size = 4-0 (for fascia)</td>
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			<td height="21" style="height:21px;">Perma-hand silk</td>
			<td>Ethicon</td>
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			<td height="21" style="height:21px;">Micro drill</td>
			<td>Roboz Surgical Instrument Co. Inc.</td>
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			<td height="21" style="height:21px;">Laser doppler flowmeter</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td>for monitoring change in blood flow</td>
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			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tool</td>
			<td>21052-00</td>
			<td>to prevent hypothermia</td>
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			<td height="21" style="height:21px;">Lab-Jack</td>
			<td>Fisher scientific&#160;</td>
			<td>14-673-50</td>
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			<td height="21" style="height:21px;">X-Y gliding stage&#160;</td>
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photothrombosis-induced focal ischemia as a model of spinal cord injury in mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

O objetivo deste estudo foi o de produzir isquemia medular em ratos usando um modelo PT. Após a região desejada do osso por cima da medula espinhal (T10 - T12) foi diluído, Rosa de Bengala foi injectado por via sinus retro-orbital, e a isquemia foi induzida pela PT A Figura 1A e B mostram o rato posicionado numa cirúrgico feito por encomenda. plataforma durante a cirurgia. O rato foi mantido no lugar por um grampo focinho e dois grampos vertebrado ajustável para estabilizar a coluna vertebral <stro...

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Fototrombose induzida por isquemia focal como um Modelo de Lesão Medular em Ratos

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

Research

JoVE Journal

Medicine

13.1K visualizações.  University of Missouri. Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice. 

Vídeo: Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Aguda e crônica teste sensorial tátil após a lesão medular em ratos

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Medicina

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Controlada por laceração do colo do útero em camundongos

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

Um modelo de contusão grave de lesão medular em ratos

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Isquemia-reperfusão modelo de lesão renal aguda e Pós Lesão fibrose em camundongos

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Reproducable Paraplegia por Thoracic Aorta Oclusão em um modelo murino de Medula Espinhal A isquemia-reperfusão

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Controlado Modelo Impact Cortical para lesão cerebral traumática

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

Um sistema de rádio-telemetria para monitorar a função cardiovascular em ratos com Medula Espinhal transecção e Embryonic Stem Neural enxertos de células-

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation em Experimental contusão Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Calibrado Fórceps Modelo de Lesão Medular Compression

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. 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 ...