Todos os procedimentos descritos são conduzidos de acordo com os Institutos Nacionais de Saúde as orientações para o cuidado e uso de animais em pesquisa e todos os protocolos de animais foram aprovadas pelo Oregon Health &amp; Science University Animal Care Institucional e Comitê de uso. 1. Preparação cirúrgica <ol><li> Pesar o mouse. O procedimento descrito é realizado em camundongos C57BL / 6 com peso entre 20 e 25 g. A anestesia é induzida em uma caixa de indução usando isoflurano 3-4% e, posteriormente, mantido usando 1,5-2,5% de isoflurano no ar / oxigênio mistura. </li><li> Lubrificar os olhos e posição supina o animal em uma almofada de aquecimento. Imobilizar quatro extremidades com fita. As patas traseiras podem ser gravadas, em uma posição neutra, no entanto, o patas dianteiras devem ser protegidos, tão perto da parede torácica quanto possível para permitir excursão parede cheia no peito durante as compressões torácicas. </li><li> Lubrificar e colocar um sensor de temperatura retal. A temperatura é controlada usando uma almofada de aquecimento e uma lâmpada ligada a um controlador eletrônico de temperatura (Digi-Sense, Cole Parmer, Vernon Hills IL), que é ajustada para manter a 37,0 ° C. Porque é possível que um gradiente de temperatura dentro do animal pode desenvolver durante o estado de ausência de fluxo, é importante que a temperatura ser medidos e controlados perto do órgão de interesse. </li><li> Intubar a traquéia usando um 2,5 cm 22 ga cateter de teflon (INSYTE-W, BD, Franklin NJ) ea extremidade cortada distal de um introdutor angular (Frova Introducer, Cook Medical, Bloomington IN). Outros métodos de intubação traqueal são aceitáveis, porém, o uso do introdutor angular permite um posicionamento adequado do pescoço em uma posição ligeiramente alargado, o que otimiza a exposição cirúrgica para a colocação do cateter intravenosa. O cubo de cateter traqueal é presa com um laço de sutura para o incisivo e mantido com uma leve tensão para imobilizar a cabeça durante as compressões torácicas. </li><li> Mecanicamente ventilar o mouse com um ventilador de roedores ajustado para 140 mL, 150 respirações / minuto. Isto não é ajustada para o peso que este protocolo é sempre realizada em 20-25 g animais. Não excesso de aprofundar a anestesia. Não defina o ventilador para aplicar pressão expiratória final como este prejudica a reanimação. </li><li> Utilizando uma técnica estéril e um microscópio cirúrgico, coloque uma pré-lavada PE-10 cateter na veia jugular. Ambos os lados podem ser usados, mas o uso do lado mais próximo ao operador reduz a chance de deslocamento do cateter durante as compressões no peito ou outras manipulações. 0,5% bupivicaine, 0,1 mL é infitrated em bordas da ferida para controlar a dor pós-operatória. </li><li> Garantir a PE-10 cateter no fechamento da pele com adesivo cirúrgico de cianoacrilato. </li><li> Colocação de eletrodos de ECG subcutâneo e se conectar ao dispositivo de monitoramento. Muita atenção ao caminho do sinal e maximização do sinal em si é fundamental para o sucesso da reanimação. Garantir que todos os fios são fixados para a superfície de funcionamento, minimizar cruzamentos de sinal, e minimizar os isoladores (como o ar em agulhas ocas ou fio flexível) dentro do percurso do sinal. Agulhas sólido ligado a cabo nonstranded pode ser comprado ou feito em laboratório. Uma vez conectado, otimizar o sinal de ECG no monitor. </li></ol> 2. Parada cardíaca <ol><li> Garantir que o mouse é normotérmica, definida como temperatura retal 36,5 º C - 37,5 ° C. Administrar 40 mL de cloreto de potássio à temperatura ambiente 0,5 M por via intravenosa e observar isoelétrico rastreamento no ECG. Ajuste da dose para o peso não é necessária dentro da faixa de 20-25 g. Iniciar o temporizador de prisão. </li><li> Desligue o ventilador. Descontinuar o vapor anestésico. Desligar o aquecimento almofada e qualquer outro equipamento que produz ruídos eletrônicos que possam interferir com a monitorização do ECG. Coloque um cobertor isolante sobre o mouse. </li><li> Temperatura recorde a cada minuto durante a parada cardíaca. Se necessário, uma lâmpada de aquecimento pode ser usado para trazer temperatura central até a faixa normotérmica. Durante a parada cardíaca, preparar material e equipamento (por exemplo, a seringa de adrenalina) para a ressuscitação. A lista pode ser útil para garantir a reanimação ininterrupto, o que é essencial para a sobrevivência. </li><li> Depois de 7 minutos e 30 segundos de parada cardíaca, volte a ligar o ventilador e aumentar a taxa de 180 ciclos / min, mantendo o volume corrente com a configuração pré-parada. </li><li> Aos 8 minutos, iniciar as compressões torácicas em 300 BPM. Artefato de movimento no ECG podem ser usados ​​para julgar taxa de CPR. Compressões torácicas devem ser entregues com o dedo indicador, 5 mm acima do apêndice xifóide e ligeiramente à esquerda da linha média. O peito deve ser comprimido 1/3-1/2 da distância ântero-posterior e recolhimento integral deve ser permitido entre as compressões. Posicionamento dos dedos e da pressão de compressão ideal são absolutamente críticos. Incapacidade de alcançar a sobrevivência neste modelo é quase sempre devido a suboptimal CPR. </li><li> Infundir 0,5 ml de adrenalina, diluída a 15 mcg / mL, nos primeiros 30 segundos de CPR. Dose de adrenalina total é de 8-12 mg. Observar atentamente o ECG para o retorno da circulação espontânea (RCE). Estreitas complexos QRS são vistos entre os artefatos de compressão. ROSC geralmente ocorre entre 90 segundos e dois minutos após o início do CPR. Ressuscitação é abandonado se ROSC não ocorre por 3 minutos. Freqüentes contrações ventriculares e alterações no eletrocardiograma eixo são observados nos primeiros 2 minutos após ROSC, e quase sempre resolver em taquicardia sinusal estável em 2 minutos. </li><li> Registro de tempo total de ressuscitação e dose de adrenalina. Temperatura recorde a cada minuto durante 10 minutos após ROSC. </li><li> Leva EKG pode ser removida quando a respiração espontânea começa, geralmente dentro de 12-15 minutos após ROSC. </li><li> Extubação da traquéia quando a freqüência respiratória espontânea é&gt; 60/min. </li><li> Posicione o mouse em uma gaiola de recuperação em uma superfície de temperatura controlada definido para 37 ° C para o primeiro pós-procedimento horas 2, garantindo acesso fácil a alimentos e água. A gaiola pode então ser movido a norma condições de moradia no pós-operatório quando o mouse está completamente recuperado da anestesia e está ativo. </li></ol> 3. Exsanguination / Perfusão Fixação e Colheita Kidney <ol><li> 24h após CA / CPR, induzir a anestesia com isoflurano 3-4% e seguro do animal em decúbito dorsal sobre uma superfície cirúrgica dentro de um exaustor que é apropriado para uso com formol .. </li><li> Aprofundar a anestesia por aumento gradual da concentração de vapor anestésico para 5% e garantir o mouse está profundamente anestesiados como evidenciado pela cessação da respiração espontânea. </li><li> Realizar uma toracotomia clamshell e desangrar o mouse por punção apical do ventrículo esquerdo cardíaco de acordo com técnicas padrão. </li><li> Através da mesma agulha, administrar 0,9% por infusão lenta. Para acclerate perfusão / exsanguinação, nick aurícula direita com uma tesoura. Realizar uma laparotomia e observar os rins. Quando são branqueados, soro fisiológico pode ser alterado para 4% de formol para fixação. Rins são então incluídos em parafina e corados com Fluoro Jade-B para a quantificação da necrose do epitélio tubular. </li></ol> 4. Resultados representativos: CA quando é induzida, pressão arterial média (MAP) e regionais do fluxo sanguíneo renal gota (RRBCF) para perto de zero e permanecerá estável até a reanimação começa (Figura 1). 24h após CA / CPR, índices séricos de função renal (uréia, uréia e creatinina) estão significativamente elevados em relação ao sham-operado animais. AST / ALT também são elevados, oferecendo evidências de isquemia de corpo inteiro profunda (Figura 2). Neutrófilos gelatinase associados lipocalina (NGAL), um indicador sensível de lesão isquêmica renal é maciçamente 24h upregulated após CA / CPR (Figura 3). Finalmente, fotomicrografias demonstram a necrose, patchy medular típico de lesão renal isquêmica com afinamento do epitélio tubular e preenchimento luminal em hematoxilina e eosina (Figura 4, painel A) e morte celular no epitélio tubular extensa medular quando corados com fluoro-jade B ( Figura 4, painel b). <img src="/files/ftp_upload/3116/3116fig1.jpg" alt="Figura 1" /> Figura 1. Parada cardíaca induz a perda instantânea de pressão de perfusão, representada aqui como a pressão arterial média (MAP), medido na artéria femoral, resultando em quase completa cessação do fluxo sangüíneo regional renal cortical (RRCBF) durante todo o período de parada cardíaca (sombreado área). Ressuscitação com compressões torácicas e retorna epinefrina MAP ao normal e RRCBF constante sobe no período pós ressuscitação. Reproduzido com permissão da 14. <img src="/files/ftp_upload/3116/3116fig2.jpg" alt="Figura 2" /> Figura 2. 24h pós-procedimento, uréia (BUN), creatinina sérica, ea extensão da morte celular tubular são significativamente elevados nos animais submetidos a CA / CPR, em comparação com os animais tratados com um procedimento simulado. CA / CPR induz um insulto pan-organismal isquêmica, aqui evidenciada pela elevação do maciço de função hepática enzimas alanina amino transferase (ALT) e aspartato aminotransferase (AST) no CA / CPR ratos em comparação com animais tratados com sham. <img src="/files/ftp_upload/3116/3116fig3.jpg" alt="Figura 3" /> Figura 3. Western blot realizado com anticorpos policlonais para gelatinase de neutrófilos associada lipocalina (NGAL), um indicador sensível de lesão isquêmica renal. Amostras de urina foram obtidas imediatamente antes (&quot;pre&quot;) e 24 horas depois (&quot;24 horas&quot;) CA / CPR em 4 animais (rotulado a, b, c, d e acima). NGAL é maciçamente upregulated em ratourina após CA / CPR. Reproduzido com permissão da 14. <img src="/files/ftp_upload/3116/3116fig4.jpg" alt="Figura 4" /> Figura 4. A) Hematoxilina e eosina de uma pequena seção de eixo hilar 24h tecido renal após CA / CPR. Há dano irregular, mas claro para medular e corticomedular túbulos com tubular entupimento. Setas apontam para túbulos danificado com núcleos, inchada picnóticos na junção córtico-medular. B) Flouro-jade B mancha de uma mesma região no mesmo animal, 24h após CA / CPR. Manchas Fluoro-jade B células necróticas verde brilhante, mostrando necrose corticomedular irregular tubular. Setas apontam para brilhantemente coradas túbulos danificada na junção córtico-medular. Essas descobertas são substancialmente similares aos achados de biópsia renal de humanos que desenvolvem AKI, e ao contrário daqueles produzidos por outros modelos animais de lesão renal aguda.

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Não há conflitos de interesse declarados.

O modelo normotérmica de parada cardíaca e ressuscitação cardiopulmonar em camundongos oferece caminhos múltiplos de avaliação em um modelo que reproduz a fisiopatologia e morfologia da causa clínica mais comum da AKI, de corpo inteiro hipoperfusão. Teste de hipóteses pode ser coadjuvado por acesso a uma panóplia de técnicas de manipulação genética e anatomia bem compreendido e caracterizado e fisiologia do rato de laboratório. Como descrito aqui, a sobrevivência em mãos ex...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Cloreto de Potássio </td><td> Abbott Medical </td><td> 06653-5 </td></tr><tr><td> Isoflurano </td><td> Abbott Medical </td><td> 05260-05 (e outros) </td></tr><tr><td> Epinefrina HCl </td><td> Múltiplo </td><td> Múltiplo </td></tr><tr><td> Digi-Sense controlador de temperatura </td><td> Cole-Palmer </td><td> EW-89000-00 </td></tr><tr><td> Angiocath IV Cath </td><td> VWR </td><td> 381134 </td></tr><tr><td> Frova introdutor das vias aéreas em ângulo </td><td> Cozinhar </td><td> G27282 </td></tr><tr><td> Ventilador MicroVent para Ratos </td><td> Harvard Apparatus </td><td> 733591 </td></tr></tbody></table>

normothermic cardiac arrest and cardiopulmonary resuscitation: a mouse model of ischemia-reperfusion injury

Lesão renal aguda (LRA) é uma comum, altamente letal, complicação de doença grave que tem uma alta mortalidade 1-4 e que é mais frequentemente causada por hipoperfusão de corpo inteiro. 5,6 reprodução bem sucedida de hipoperfusão de corpo inteiro em modelos de roedores foi repleto de dificuldades. 7-9,9,10 Modelos que empregam isquemia focal têm repetidamente demonstrado resultados que não se traduzem em ambiente clínico, e modelos maiores animais que permitem a toda a falta de acesso corpo hipoperfusão para o conjunto de ferramentas completo de manipulação genética possível no mouse. 11,12 No entanto, nos últimos anos um modelo do rato de parada cardíaca e reanimação cardiopulmonar surgiu o que pode ser adaptado para o modelo de LRA. 13 Este modelo reproduz de forma confiável fisiológicas, os resultados funcionais, anatômicos e histológicos observados em clínica AKI , é rapidamente repetitivo, e oferece todas as vantagens de um modelo murino cirúrgico, incluindo o acesso a técnicas de manipulação genética, relativo baixo custo para grandes animais, e facilidade de uso. Nosso grupo tem desenvolvido uma vasta experiência com o uso deste modelo para avaliar uma série de órgãos específicos resultados em AKI 14,15.

Watch this Scientific Journal Video about Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury at JoVE.com

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Um modelo poderoso para perioperatório e crítica lesão renal aguda relacionada com o cuidado é apresentado. Usando hipoperfusão corpo inteiro induzida por parada cardíaca é possível a quase replicar as alterações histológicas e funcionais do AKI clínico.

Cardiac Arrest normotérmica e Ressuscitação Cardiopulmonar: um modelo do rato de isquemia-reperfusão

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

normothermic-cardiac-arrest-cardiopulmonary-resuscitation-mouse-model

Research

JoVE Journal

Medicine

17.9K visualizações.  Oregon Health & Sciences University. Um modelo poderoso para perioperatório e crítica lesão renal aguda relacionada com o cuidado é apresentado. Usando hipoperfusão corpo inteiro induzida por parada cardíaca é possível a quase replicar as alterações histológicas e funcionais do AKI clínico.

Vídeo: Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

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 ...

Medicina

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Indução e Avaliação de isquemia-reperfusão em Langendorff-perfundidos Rato Corações

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Modelo murino de isquemia-reperfusão

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Um modelo de mouse Retinal isquemia-reperfusão Através elevação da pressão intra-ocular

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Ultrasonographic Assessment During Cardiopulmonary Resuscitation

The US-CAB (Ultrasound, Circulation/Airway/Breathing) protocol integrates several sonographic techniques into a structured assessment of the circulation, airway, and breathing status of a patient during cardiopulmonary resuscitation (CPR) in an advanced life support-compliant manner. US-C provides a subxiphoid view of the heart, to look for potentially reversible causes of disease, such as pericardial effusion, pulmonary embolism, hypovolemia, and acute coronary thrombosis. Sonographic cardiac activity during CPR not only helps differentiate pseudo-pulseless electrical activity (PEA) from true PEA but also represents a higher chance of the return of spontaneous circulation (ROSC) and survival. Evaluation of the inferior vena cava (IVC) shows the fluid status of the patient and indicates the best methods to use for fluid resuscitation. If aortic dissection is suspected, a subxiphoid view of the aorta is suggested for identifying an intimal flap. Once intubation is done, tracheal ultrasound (US-A) at the suprasternal notch helps differentiate endotracheal intubation (one air-mucosal interface with one comet-tail) from esophageal intubation (double tract sign). Immediately following US-A, bilateral lung US (US-B) should be done to confirm proper bilateral ventilation using the lung sliding sign. In addition, US-C can be serially followed to see the dynamic changes in the cardiac chambers and IVC, or any cardiac contraction suggestive of ROSC. US-B can also detect coexisting lung or pleural pathologies without interfering with the performance of CPR. The main concern when implementing this method is maintaining high-quality CPR without delays in chest compressions when performing US-CAB. Rigorous training and continued practice are key to minimize any interruptions during resuscitation.

Here, we present a US-CAB (Ultrasound, Circulation/Airway/Breathing) protocol for use during cardiopulmonary resuscitation (CPR). US-C evaluates the subxiphoid view of the heart and inferior vena cava. After intubation, tracheal US (US-A) and lung US (US-B) help confirm endotracheal intubation and proper ventilation.

Avaliação ultrasonográfica durante a ressuscitação cardiopulmonar

The US-CAB (Ultrasound, Circulation/Airway/Breathing) protocol integrates several sonographic techniques into a structured assessment of the circulation, ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Um modelo pré-clínico do rato para o estudo da lesão da Isquemia-reperfusion na microcirurgia reconstrutiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

Entrega intramiocárdica atrasada de células-tronco após lesão de reperfusão de Isquemia em um modelo murino

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Modelo de Lesão de Isquemia/Reperfusão de Pulmão de Rato em Transplante de Pulmão de Rato: Otimizações para Melhorar os Resultados

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Um modelo eficaz de rato de isquemia renal unilateral-reperfusão lesão

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

A Piglet Perinatal Asphyxia Model to Study Cardiac Injury and Hemodynamics after Cardiac Arrest, Resuscitation, and the Return of Spontaneous Circulation

Neonatal piglets have been extensively used as translational models for perinatal asphyxia. In 2007, we adapted a well-established piglet asphyxia model by introducing cardiac arrest. This enabled us to study the impact of severe asphyxia on key outcomes, including the time taken for the return of spontaneous circulation (ROSC), as well as the effect of chest compressions according to alternative protocols for cardiopulmonary resuscitation. Due to the anatomical and physiological similarities between piglets and human neonates, piglets serve as good models in studies of cardiopulmonary resuscitation and hemodynamic monitoring. In fact, this cardiac arrest model has provided evidence for guideline development through research on resuscitation protocols, pathophysiology, biomarkers, and novel methods for hemodynamic monitoring. Notably, the incidental finding that a substantial fraction of piglets have pulseless electrical activity (PEA) during cardiac arrest may increase the applicability of the model (i.e., it may be used to study pathophysiology extending beyond the perinatal period). However, the model generation is technically challenging and requires various skill sets, dedicated personnel, and a fine balance of the measures, including the surgical protocols and the use of sedatives/analgesics, to ensure a reasonable rate of survival. In this paper, the protocol is described in detail, as well as experiences with adaptations to the protocol over the years.

This piglet model involves surgical instrumentation, asphyxiation until the cardiac arrest, resuscitation, and post-resuscitation observation. The model allows for multiple sampling per animal, and by using continuous invasive arterial blood pressure, ECG, and non-invasive cardiac output monitoring, it provides knowledge about hemodynamics and cardiac pathophysiology in perinatal asphyxia and neonatal cardiopulmonary resuscitation.

Modelo de Asfixia Perinatal de Leitões para Estudo da Lesão Cardíaca e Hemodinâmica após Parada Cardíaca, Ressuscitação e Retorno à Circulação Espontânea

Neonatal piglets have been extensively used as translational models for perinatal asphyxia. In 2007, we adapted a well-established piglet asphyxia model ...