Name: photothrombosis-induced focal ischemia as a model of spinal cord injury in mice
Uploaded: 2015-07-16T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

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.

<table><tbody><tr><td>Rose Bengal</td><td>Sigma-Aldrich</td><td>330000</td><td>20 mg/ml in sterile saline</td></tr><tr><td>C57BL/6J</td><td>Jackson lab</td><td>664</td><td>22 - 25 g</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-996-06</td><td>100 mg/ml</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-234-11</td><td>100 mg/ml</td></tr><tr><td>Betadine solution</td><td>Purdue</td><td>NDC-67618-150-01</td><td>10% povidone iodine topical solution</td></tr><tr><td>Normal saline</td><td>Abott Laboratories</td><td>04930-04-10</td><td>For diluting RB, anaesthesia and for preventing tissue from drying</td></tr><tr><td>Artificial tears ointment&amp;nbsp;</td><td>Rugby</td><td>NDC-0536-6550-91</td><td>83% white petrolatum</td></tr><tr><td>Ethanol</td><td>Decon labs.Inc</td><td>2716</td><td>70% ethanol for disinfection</td></tr><tr><td>Metal halide lamp</td><td>EXFO, Canada</td><td>X-Cite 120 PC&amp;nbsp;</td><td>Set power at 12%</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tool</td><td>15000-10</td><td>for minor dissection</td></tr><tr><td>Scissors (angled to side)</td><td>Fine Science Tool</td><td>14063-011</td><td>No. 3 handle</td></tr><tr><td>Standard scalpel</td><td>Fine Science Tool</td><td>10003-12</td><td>for removing muscle</td></tr><tr><td>Scalpel blade</td><td>Feather</td><td>2976</td><td>No. 10</td></tr><tr><td>Forceps (curved)</td><td>Fine Science Tool</td><td>11150-10</td><td>for holding tissue</td></tr><tr><td>Forceps (straight)</td><td>Fine Science Tool</td><td>11151-10</td><td>for holding tissue</td></tr><tr><td>Needle holder&amp;nbsp;</td><td>Fine Science Tool</td><td>12002-12</td><td>for suturing</td></tr><tr><td>Tissue adhesive glue</td><td>3M Vetbond</td><td>1469SB</td><td>to adhere to edges of the cut skin</td></tr><tr><td>Monofilament polypropylene&amp;nbsp;</td><td>USSC Sutures</td><td>VP-521</td><td>Size = 4-0 (for fascia)</td></tr><tr><td>Perma-hand silk</td><td>Ethicon</td><td>683G</td><td>Size = 4-0 (for skin)</td></tr><tr><td>Micro drill</td><td>Roboz Surgical Instrument Co. Inc.</td><td>RS-6300</td><td>with bone polishing drill bit</td></tr><tr><td>Laser doppler flowmeter</td><td>Moor Instruments</td><td>moorVMS-LDF1</td><td>for monitoring change in blood flow</td></tr><tr><td>Heating pad</td><td>Fine Science Tool</td><td>21052-00</td><td>to prevent hypothermia</td></tr><tr><td>Lab-Jack</td><td>Fisher scientific&amp;nbsp;</td><td>14-673-50</td><td>4 x 4 in plate to adjust the height of the animal</td></tr><tr><td>X-Y gliding stage&amp;nbsp;</td><td>Amscope</td><td>GT100</td><td>for positioning the animal under microscope&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

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

13.3K 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

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Isquemia Photothrombotic: Um Modelo de lesão cortical fotoquímica Minimamente Invasiva e reprodutível para mouse Estudos curso

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Medicina

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 ...

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 ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Um Modelo de Lesão Medular Contusiva Baseado em Cilindrada de Tecidos em Ratos

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Indução da lesão da medula espinhal tipo transssecção completa em camundongos

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

Neurociência

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Modelagem de AVC em Camundongos: Lesões Corticais Focais por Fototrombose

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Estabelecendo um Modelo de Lesão da Medula Espinhal por Contusão de Camundongo Baseado em uma Técnica Minimamente Invasiva

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

Estabelecimento do Modelo de Síndrome do Cordão Central em Camundongos C57BL/6J

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

Source: Llovera, G. et al. <a href="https://www.jove.com/v/62536">Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to generate photothrombotic stroke in a mouse model by laser illumination of an intact skull following administration of photosensitive dye.

Modelagem de AVC Fototrombótico em Modelo de Camundongo: Uma Técnica de Iluminação a Laser Através do Crânio de Camundongo Após Administração de Corante Fotossensível para Induzir Fototrombose

Source: Llovera, G. et al. Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis. J. Vis. Exp. (2021)
This video demonstrates the procedure ...

JoVE Encyclopedia of Experiments

Biologia

Modelo de Roedores

Adulto

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

<a href='https://app.jove.com/v/64538/establishing-mouse-contusion-spinal-cord-injury-model-based-on'>Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp.&#160;(2022).</a>This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.