Name: a preclinical model of exertional heat stroke in mice
Uploaded: 2021-07-01T14:30:00
Description: Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

Este trabalho foi financiado pelo Departamento de Defesa W81XWH-15-2-0038 (TLC) e BA180078 (TLC) e pelo BK e Betty Stevens Endowment (TLC). A JMA foi apoiada pela ajuda financeira do Reino da Arábia Saudita. Michelle King estava na Universidade da Flórida no momento em que este estudo foi conduzido. Atualmente trabalha no Gatorade Sports Science Institute, uma divisão da PepsiCo P&D.

Todos os procedimentos foram revisados e aprovados pela Universidade da Flórida IACUC. C57BL/6J camundongos machos ou fêmeas, ~4 meses de idade, pesando dentro de uma faixa de 27-34 g e 20-25 g, respectivamente, são usados para o estudo.
1. Implantação cirúrgica do sistema de monitoramento de temperatura telemétrica
<ol><li>Ao chegar do vendedor, deixe os animais descansarem no vivarium por pelo menos 1 semana antes da cirurgia para minimizar o estresse do transporte.</li>
	<li>Grupo ...

<ol>
	<li>Leon, L. R., Bouchama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stroke.">Heat stroke.</a> Comprehensive Physiology. 5 (2), 611-647 (2015).</li><li>Laitano, O., Leon, L. R., Roberts, W. O., Sawka, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controversies+in+exertional+heat+stroke+diagnosis,+prevention,+and+treatment.">Controversies in exertional heat stroke diagnosis, prevention, and treatment.</a> Journal of Applied Physiology. 127 (5), 1338-1348 (2019).</li><li>King, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+prior+illness+on+exertional+heat+stroke+presentation+and+outcome.">Influence of prior illness on exertional heat stroke presentation and outcome.</a> PLOS One. 14 (8), 0221329 (2019).</li><li>Carter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+hospitalizations+and+deaths+from+heat+illness+in+soldiers.">Epidemiology of hospitalizations and deaths from heat illness in soldiers.</a> Medicine and Science in Sports and Exercise. 37 (8), 1338-1344 (2005).</li><li>Howe, A. S., Boden, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-related+illness+in+athletes.">Heat-related illness in athletes.</a> The American Journal of Sports Medicine. 35 (8), 1384-1395 (2007).</li><li>Wallace, R. F., Kriebel, D., Punnett, L., Wegman, D. H., Amoroso, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prior+heat+illness+hospitalization+and+risk+of+early+death.">Prior heat illness hospitalization and risk of early death.</a> Environmental Research. 104 (2), 290-295 (2007).</li><li>Wang, J. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+heat+stroke+and+subsequent+cardiovascular+diseases.">The association between heat stroke and subsequent cardiovascular diseases.</a> PLOS One. 14 (2), 0211386 (2019).</li><li>Leon, L. R., Blaha, M. D., DuBose, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+cytokine,+corticosterone,+and+tissue+injury+responses+in+mice+during+heat+strain+recovery.">Time course of cytokine, corticosterone, and tissue injury responses in mice during heat strain recovery.</a> Journal of Applied Physiology. 100 (4), 1400-1409 (2006).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 288 (1), 197-204 (2005).</li><li>Leon, L. R., Gordon, C. J., Helwig, B. G., Rufolo, D. M., Blaha, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoregulatory,+behavioral,+and+metabolic+responses+to+heatstroke+in+a+conscious+mouse+model.">Thermoregulatory, behavioral, and metabolic responses to heatstroke in a conscious mouse model.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 299 (1), 241-248 (2010).</li><li>King, M. A., Leon, L. R., Morse, D. A., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+cytokine+and+chemokine+responses+to+exertional+heat+stroke+in+mice.">Unique cytokine and chemokine responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 122 (2), 296-306 (2016).</li><li>Costa, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=l-Arginine+supplementation+prevents+increases+in+intestinal+permeability+and+bacterial+translocation+in+Male+Swiss+mice+subjected+to+physical+exercise+under+environmental+heat+stress.">l-Arginine supplementation prevents increases in intestinal permeability and bacterial translocation in Male Swiss mice subjected to physical exercise under environmental heat stress.</a> The Journal of Nutrition. 144 (2), 218-223 (2014).</li><li>Hubbard, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+in+the+heat+on+predisposition+to+heatstroke.">Effects of exercise in the heat on predisposition to heatstroke.</a> Medicine and Science in Sports. 11 (1), 66-71 (1979).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+acute+heatstroke+mortality.">Rat model of acute heatstroke mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 42 (6), 809-816 (1977).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+significance+of+selected+serum+enzymes+in+a+rat+heatstroke+model.">Diagnostic significance of selected serum enzymes in a rat heatstroke model.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 46 (2), 334-339 (1979).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+physical+effort+in+the+etiology+of+rat+heatstroke+injury+and+mortality.">Role of physical effort in the etiology of rat heatstroke injury and mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 45 (3), 463-468 (1978).</li><li>Garcia, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-dependent+responses+to+exertional+heat+stroke+in+mice.">Sex-dependent responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 125 (3), 841-849 (2018).</li><li>Garcia, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ibuprofen+during+Exertional+Heat+Stroke+in+Mice.">Effects of Ibuprofen during Exertional Heat Stroke in Mice.</a> Medicine and Science in Sports and Exercise. 52 (9), 1870-1878 (2020).</li><li>King, M. A., Leon, L. R., Mustico, D. L., Haines, J. M., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+multi-organ+injury+in+a+pre-clinical+model+of+exertional+heat+stroke.">Biomarkers of multi-organ injury in a pre-clinical model of exertional heat stroke.</a> Journal of Applied Physiology. 118 (10), (2015).</li><li>Murray, K. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exertional+heat+stroke+leads+to+concurrent+long-term+epigenetic+memory,+immunosuppression+and+altered+heat+shock+response+in+female+mice.">Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.</a> The Journal of Physiology. 599 (1), 119-141 (2021).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 288, 197-204 (2005).</li><li>Laitano, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+metabolic+dysfunction+in+myocardium+following+exertional+heat+stroke+in+mice.">Delayed metabolic dysfunction in myocardium following exertional heat stroke in mice.</a> The Journal of Physiology. 598 (5), 967-985 (2020).</li><li>Iwaniec, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+phase+response+to+exertional+heat+stroke+in+mice.">Acute phase response to exertional heat stroke in mice.</a> Experimental Physiology. 106 (1), 222-232 (2020).</li><li>He, S. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+rhabdomyolysis+model+in+mice+with+exertional+heat+stroke+mouse+model+of+EHS-rhabdomyolysis.">Optimization of a rhabdomyolysis model in mice with exertional heat stroke mouse model of EHS-rhabdomyolysis.</a> Frontiers in Physiology. 11, (2020).</li><li>Lopez, J. R., Kaura, V., Diggle, C. P., Hopkins, P. M., Allen, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+hyperthermia,+environmental+heat+stress,+and+intracellular+calcium+dysregulation+in+a+mouse+model+expressing+the+p.G2435R+variant+of+RYR1.">Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1.</a> British Journal of Anaesthesia. 121 (4), 953-961 (2018).</li><li>Laitano, O., Murray, K. O., Leon, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+mechanisms+of+exertional+heat+stroke+and+malignant+hyperthermia:+evidence+vs.+conjecture.">Overlapping mechanisms of exertional heat stroke and malignant hyperthermia: evidence vs. conjecture.</a> Sports Medicine. 50 (9), 115-123 (2020).</li><li>Casa, D. J., Armstrong, L. E., Kenny, G. P., O'Connor, F. G., Huggins, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exertional+heat+stroke:+new+concepts+regarding+cause+and+care.">Exertional heat stroke: new concepts regarding cause and care.</a> Current Sports Medicine Reports. 11 (3), 115-123 (2012).</li></ol>

Esta revisão técnica visa fornecer diretrizes para o desempenho de um modelo pré-clínico de EHS em camundongos. São fornecidas etapas e materiais detalhados necessários para a execução de um episódio de EHS reprodutível de gravidades variáveis. É importante ressaltar que o modelo imita em grande parte os sinais, sintomas e disfunção multi-órgãos observados em vítimas humanas de EHS11,19. Além disso, este modelo permite o exame do mecanismo subja...

A insolação é caracterizada por disfunção do sistema nervoso central e danos subsequentes aos órgãos em indivíduos hipertérmicos1. Há duas manifestações de insolação. A insolação clássica (CHS) afeta principalmente populações idosas durante ondas de calor ou crianças deixadas em veículos expostos ao sol durante os dias quentes de verão1. O insolação exercional (EHS) ocorre quando há uma incapacidade de termregular adequadamente durante o esforço físico, tipicamente, mas nem sempre, sob altas temperaturas ambientais resultando em sintomas neurológicos, hipertermia e subsequente disfunção multi-órgãos e danos2. EHS ocorre em atletas recreativos e de elite, bem como militares e trabalhadores com e sem desidratação concomitante3,4. De fato, a EHS é a terceira principal causa de mortalidade em atletas durante a atividade física5. É extremamente desafiador estudar eHS em humanos, pois o episódio pode ser letal ou levar a desfechos negativos de saúde a longo prazo6,7. Portanto, um modelo pré-clínico confiável de EHS poderia servir como uma ferramenta valiosa para superar as limitações das observações clínicas retrospectivas e associativas em vítimas humanas de EHS. Modelos pré-clínicos de CHS em roedores e suínos têm sido bem caracterizados8,9,10. No entanto, modelos pré-clínicos de CHS não se traduzem diretamente na fisiopatologia EHS devido aos efeitos únicos do exercício físico no perfil termoregulatório e resposta imune inata11. Além disso, tentativas anteriores de desenvolver modelos de EHS pré-clínicos em roedores apresentaram restrições significativas, incluindo estímulos de estresse sobrepostos induzidos por choque elétrico, inserção de uma sonda retatal e temperaturas corporais máximas predefinidas com altas taxas de mortalidade12,13,14,15,16 que não correspondem aos dados epidemiológicos atuais. Isso representa limitações significativas que podem confundir a interpretação dos dados e fornecer índices biomarcadores não confiáveis. Portanto, o protocolo visa caracterizar e descrever os passos de um modelo préclínico padronizado, altamente repetível e translacionável de EHS em camundongos que está em grande parte livre das limitações mencionadas acima. São descritos ajustes no modelo que podem resultar em desfechos fisiológicos classificados de insolação moderada a fatal. Para o conhecimento dos autores, este é o único modelo pré-clínico da EHS com tais características, possibilitando a busca de pesquisas relevantes de EHS de forma orientada a hipóteses11,17,18.

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a preclinical model of exertional heat stroke in mice

O insolação é a manifestação mais severa de doenças relacionadas ao calor. A insolação clássica (CHS), também conhecida como insolação passiva, ocorre em repouso, enquanto a insolação exercional (EHS) ocorre durante a atividade física. EHS difere do CHS em etiologia, apresentação clínica e sequelas de disfunção multi-órgãos. Até recentemente, apenas modelos de CHS foram bem estabelecidos. Este protocolo visa fornecer diretrizes para um modelo de mouse pré-clínico refinado de EHS que esteja livre de fatores limitantes importantes, como o uso de anestesia, contenção, sondas retais ou choque elétrico. Camundongos C57Bl/6 machos e fêmeas, instrumentados com sondas telemétricas de temperatura do núcleo (Tc) foram utilizados neste modelo. Para a familiarização com o modo de corrida, os ratos passam por 3 semanas de treinamento usando rodas de corrida voluntárias e forçadas. Depois disso, os ratos correm em uma roda forçada dentro de uma câmara climática fixada em 37,5 °C e 40%-50% de umidade relativa (RH) até apresentar limitação dos sintomas (por exemplo, perda de consciência) em Tc de 42,1-42,5 °C, embora os resultados adequados possam ser obtidos a temperaturas de câmara entre 34,5-39,5 °C e umidade entre 30%-90%. Dependendo da gravidade desejada, os camundongos são removidos da câmara imediatamente para recuperação em temperatura ambiente ou permanecem na câmara aquecida por uma duração mais longa, induzindo uma exposição mais grave e uma maior incidência de mortalidade. Os resultados são comparados com controles de exercício (EXC) e/ou controles ingênuos (NC). O modelo espelha muitos dos desfechos fisiofisiológicos observados na EHS humana, incluindo perda de consciência, hipertermia grave, danos multi-órgãos, bem como liberação inflamatória de citocinas, e respostas de fase aguda do sistema imunológico. Este modelo é ideal para pesquisas orientadas a hipóteses para testar estratégias preventivas e terapêuticas que possam retardar o surgimento da EHS ou reduzir o dano multi-órgão que caracteriza essa manifestação.

Os perfis termoregulatórios típicos durante a totalidade do protocolo EHS e a recuperação antecipada de um mouse são ilustrados na Figura 1A. Este perfil compreende quatro fases distintas que podem ser definidas como o estágio de aquecimento da câmara, estágio de exercício incremental, estágio de exercício de estado estável e um estágio de recuperação por um método de resfriamento rápido (R) ou grave (S)17. Os principais desfechos termoregulatórios i...

Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

A Preclinical Model of Exertional Heat Stroke in Mice

O protocolo descreve o desenvolvimento de um modelo padronizado, repetível e pré-clínico de insolação exercional (EHS) em camundongos livres de estímulos externos adversos, como choque elétrico. O modelo fornece uma plataforma para estudos mecanicistas, preventivos e terapêuticos.

Um modelo pré-clínico de insolação exercional em camundongos

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

Anteriormente, apenas modelos passivos de insolação foram bem estabelecidos. Este modelo de insolação exercional é livre de grandes limitações e imita mais de perto a fisiopatologia humana. Não é possível estudar efetivamente a insolação em humanos porque é uma condição de risco de vida.Este modelo pré-clínico mantém as características clínicas da insolação do esforço humano. Esta técnica ajuda a testar os efeitos da taxa de resfriamento na recuperação de insolação exercional, comorbidades da ingestão de drogas, bem como consequências a longo prazo da exposição ao insolação. Demonstrando o procedimento estará Jamal Alzahrani, um estudante sênior de doutorado do laboratório do Dr. Clanton.Depois de anestesiar o camundongo, use lubrificante ocular, como a Pomada Veterinária para proteger os olhos do animal de danos ou lesões durante a cirurgia. Para preparar o local cirúrgico, raspe o abdômen inferior com pequenos cortadores de cabelo animal ou um removedor de cabelo comercialmente disponível. Administre a primeira dose de 0,1 miligramas subcutâneos por quilograma de buprenorfina.Esfregue a área com três lavagens de povidone-iodo ou clorhexidina, seguido de enxaguar álcool isopropílico de 70%, depois transfira o rato para a área cirúrgica, use uma cortina adesiva para isolar o local cirúrgico no camundongo. Usando instrumentos estéreis na técnica asséptica, faça uma incisão de aproximadamente um centímetro na linha média ao longo da linha alba cerca de 0,5 centímetros da margem costeira, em seguida, separe a pele da camada muscular e faça uma incisão ligeiramente menor na linha alba tomando cuidado para não danificar o intestino ou órgãos internos. Uma vez que a camada muscular esteja aberta, coloque o telemetro estéril na cavidade intraperitoneal na frente das artérias e veias caudais e dorsal aos órgãos digestivos para permitir que ele flutue livremente.Feche a abertura abdominal com uma sutura absorvível de 5-O estéril e feche a pele usando um simples ponto interrompido com sutura de prolina 5-O. Coloque o mouse em sua gaiola limpa com uma almofada de aquecimento de micro-ondas aconchegante sob a gaiola. Monitore o rato a cada 15 minutos durante a primeira hora de recuperação da anestesia e depois retorne ao centro de alojamento de animais.Na noite anterior ao protocolo de insolação exercional, coloque o rato na câmara ambiental à temperatura ambiente para se aclimatar à câmara. Use o sistema de aquisição de dados para coletar temperatura contínua do núcleo ou TC em média mais de intervalos de 30 segundos durante a noite. Na manhã do protocolo de insolação exercional, certifique-se de que o rato esteja na faixa normal de temperatura diurna de 36 a 37,5 graus Celsius antes de aumentar a temperatura da câmara para garantir que o rato não tenha febre ou tenha experimentado estresse indevido durante este período.Uma vez que o rato esteja estável e dentro de uma faixa de temperatura normal do núcleo de repouso, remova a comida e a água e pese o animal. Feche a porta da câmara e aumente a temperatura da câmara para uma meta de 37,5 graus Celsius e 40 a 50% de umidade relativa ou a temperatura e umidade ambiental desejadas. Verifique a temperatura e a umidade da câmara com um monitor de temperatura e umidade calibrado.Cerque a câmara com uma cortina de apagão para manter a luz e os distúrbios mínimos durante o protocolo. Monitore o mouse continuamente durante o protocolo através de câmeras remotas iluminadas por RI. Concentre a segunda câmera no monitor de temperatura e umidade colocado perto da roda de corrida.Faça ajustes no controlador para o ponto de ajuste da câmara ambiental para garantir leituras precisas de temperatura perto do animal. Uma vez que a câmara tenha atingido sua temperatura alvo medida pela segunda câmera no monitor de temperatura, abra rapidamente a porta da câmara e coloque o mouse na roda de corrida forçada. Inicie o protocolo de roda de corrida forçada a uma velocidade de 2,5 metros por minuto, e aumente a velocidade em 0,3 metros por minuto a cada 10 minutos até que o mouse atinja uma temperatura central de 41 graus Celsius.Uma vez alcançada a temperatura do núcleo, permita que a velocidade permaneça constante até que ocorra a limitação dos sintomas, caracterizada por uma aparente perda de consciência, uma queda ou desmaio para trás, e a incapacidade de continuar correndo ou segurando a roda. Confirme o ponto de tempo quando o mouse tiver três rotações para trás no volante sem sinais de resposta física. Alternativamente, identifique o ponto final humano seguindo as regras locais da IACUC para determinar quando parar o protocolo.Este ponto final é ligeiramente acima da limitação dos sintomas em essencialmente todos os camundongos. Para executar o protocolo de resfriamento rápido, pare a roda assim que o mouse atingir a limitação dos sintomas e remova o mouse imediatamente da roda de corrida forçada. Pesar o rato e colocá-lo de volta em sua gaiola para se recuperar à temperatura ambiente.Durante esse tempo, deixe a porta da câmara aberta e retorne o ponto de configuração da incubadora à temperatura ambiente para permitir que a câmara esfrie rapidamente, o que resulta em uma sobrevida superior a 99% a longo prazo. Para realizar uma exposição mais severa ao insolação de insolação, mantenha a gaiola do animal dentro da câmara de 37,5 graus Celsius durante o protocolo de insolação exercional. Quando o animal atingir a limitação dos sintomas, permita que eles permaneçam na roda de corrida até que retornem à consciência como observado pela câmera remota, então remova rapidamente o mouse da roda de corrida e devolva-o diretamente à sua gaiola pré-aquecida para resultar em um perfil de resfriamento muito mais lento, essencialmente eliminando a fase hipotérmica do insolação.Remova a parte superior do filtro da gaiola durante este período para melhorar o equilíbrio com a câmara. Uma gaiola de recuperação pré-resfriada à temperatura ambiente pode ser usada para realizar um procedimento alternativo menos grave para resultar em uma fase hipotérmica suprimida com uma taxa de sobrevivência de 100% Para o protocolo de insolação exercional grave, monitore cuidadosamente os camundongos durante a recuperação e verifique continuamente os pontos finais humanos, incluindo movimentos normais durante a recuperação, como o preparo, respiração normal, e lambendo. Monitore a temperatura do núcleo durante este tempo.É improvável que os camundongos se recuperem se a temperatura do núcleo reverter a direção durante a fase de recuperação, eventualmente excedendo 40 graus Celsius. Nesse caso, encerre o experimento e continue avaliando o mouse para pontos finais humanos padrão. Os perfis termoregulatórios típicos durante a totalidade do protocolo de insolação exercional na recuperação precoce de um rato compreendem quatro fases distintas que podem ser definidas como o estágio de aquecimento da câmara, estágio de exercício incremental, estágio de exercício de estado estável e um estágio de recuperação por um método de resfriamento rápido ou lento.Para o modelo severo, o perfil de temperatura de recuperação indicou que a temperatura do núcleo permaneceu acima de 37 graus Celsius durante o período de recuperação de duas horas. Camundongos fêmeas eram mais resistentes à insolação neste modelo e corriam quase duas vezes mais distâncias do que os camundongos machos. Não foi observada diferença no tempo necessário para se recuperar a 39,5 graus Celsius nos dois modelos.No entanto, o tempo para esfriar até a temperatura ambiental de 37,5 graus Celsius foi bastante prolongado. O mais importante é o monitoramento cuidadoso dos animais em todos os momentos pós-cirurgia e insolação pós-esforço. Este método ajudou a estudar os efeitos a longo prazo da exposição ao insolação por até vários meses, e também nos permitiu medir os efeitos de fatores adicionais na doença do insolação, como a ingestão de drogas anti-inflamatórias não esteroides e epigenética.

a-preclinical-model-of-exertional-heat-stroke-in-mice

Research

JoVE Journal

Medicine

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

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

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

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

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

Medicina

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Transferência de genes para insuficiência cardíaca isquêmica em um modelo pré-clínica

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Intraluminal artéria cerebral média Oclusão Model (MCAO) para Acidente Vascular Cerebral Isquêmico com Laser Doppler Fluxometria Orientação em Ratos

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Um modelo murino de hemorragia subaracnóide

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Um Modelo murino de metástases hepáticas pré-clínica

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Curso de modelagem em Ratos: Coagulação permanente da artéria distal Cerebral Média

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

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

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

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

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

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Abordagem cirúrgica para oclusão da artéria cerebral média e reperfusão induzida por acidente vascular cerebral em ratos

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

Imagem de Neuroinflammation usando [11C] DPA-713 em um modelo do rato de acidente vascular cerebral isquêmico do animal de estimação

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...