Name: methods for acute and subacute murine hindlimb ischemia
Uploaded: 2016-06-21T15:00:00
Description: Watch this Scientific Journal Video about Methods for Acute and Subacute Murine Hindlimb Ischemia at JoVE.com

This study was supported by NIH grants R21HL118661, R56HL124444, and R01HL124444 to CDK, and by NIH grants R00HL103797 and R01HL125695 to JMM.

Todos os experimentos com animais foram realizados de acordo com o protocolo aprovado pelo Comitê de Cuidado e Uso do Duque Institucional Animal. ratos machos foram utilizados neste estudo, apesar de ambos os sexos, pode ser utilizado como indicado para o objectivo científico do estudo. 1. Remoção do cabelo <ol><li> Antes da indução da anestesia, configurar uma área de preparação de pré-cirúrgico constituído por uma almofada de aquecimento coberto fixada em 37 ° C e uma porta ...

<ol>
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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=A+quantitative+trait+locus+(LSq-1)+on+mouse+chromosome+7+is+linked+to+the+absence+of+tissue+loss+after+surgical+hindlimb+ischemia.">A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.</a> Circulation. 117, 1207-1215 (2008).</li><li>Rivard, 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=Age-dependent+impairment+of+angiogenesis.">Age-dependent impairment of angiogenesis.</a> Circulation. 99, 111-120 (1999).</li><li>Hazarika, S., 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=Impaired+angiogenesis+after+hindlimb+ischemia+in+type+2+diabetes+mellitus:+differential+regulation+of+vascular+endothelial+growth+factor+receptor+1+and+soluble+vascular+endothelial+growth+factor+receptor+1.">Impaired angiogenesis after hindlimb ischemia in type 2 diabetes mellitus: differential regulation of vascular endothelial growth factor receptor 1 and soluble vascular endothelial growth factor receptor 1.</a> Circ. 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Talvez o passo mais desafiadora no presente processo é a separação da artéria femoral a partir da veia femoral. O maior diâmetro e mais finas paredes da veia femoral em comparação com aqueles da artéria aumentar a sua susceptibilidade para perfurar e rasgar durante a manipulação cirúrgica. A probabilidade de perturbar a veia pode ser reduzido, mantendo a ferida húmida utilizando uma zaragatoa estéril humedecida com PBS. É também importante para garantir que todos os fórceps são afiadas, alinhados, e liv...

Doença arterial periférica (DAP) é uma das principais causas de morbidade e mortalidade cardiovascular em países desenvolvidos 1. resultados Pad de obstrução aterosclerótica das artérias periféricas, que leva a isquemia de membros com a dor de esforço ou de descanso resultante e, ocasionalmente, úlceras e gangrena que necessitam de amputação do membro não-cura. Terapias enfocadas PAD são essencialmente direccionado para endovascular 2 ou revascularização cirúrgica 3, como essencialmente não existem terapias médicas eficazes 4. Infelizmente, a revascularização é muitas vezes de benefício limitado, como enxertos têm elevadas taxas de insucesso (até 50% dentro de 5 anos) 5 que são piores em algumas populações (por exemplo, fumantes, mulheres, enxertos de veia safena não) 6,7. abordagens endovasculares, como a angioplastia e colocação de stent, também estão comprometidos por elevadas taxas de restenose (em excesso de 50% a 1 ano), especialmente in femoropoplítea doença 8, embora o uso de balões e stents farmacológicos melhorou resultados pouco 9-11. A fim de desenvolver novos tratamentos para a DAP é essencial para desenvolver modelos animais que reproduzem de forma fiável a doença humana. Até à data, o modelo mais comum de DAP é o modelo da pata traseira isquemia (HLI), que é mais frequentemente realizada em ratos 12,13. Na sua manifestação mais comum, o modelo envolve a ligadura cirúrgica da artéria femoral proximal e distai e os seus intervenientes ramos laterais seguida por excisão do vaso, resultando na oclusão do fluxo sanguíneo e a indução de isquemia aguda. HLI tem sido principalmente utilizado para estudar as respostas angiogénicas e arteriogénica no tecido muscular do membro periférico e os efeitos de várias terapias (por exemplo, drogas, a entrega do gene, as células estaminais) sobre essas respostas. Mais recentemente, o nosso grupo utilizou este modelo para examinar o papel das células do músculo esquelético in a resposta à isquemia e os efeitos das diferenças genéticas sobre os resultados 14. O modelo HLI tem facilitado a nossa compreensão actual que as respostas vasculares e dos músculos a isquemia são dependentes genética (isto é, estirpe consanguínea) 15, 16 anos de idade, e a presença ou ausência de outras doenças ou condições relevantes para a aterosclerose, incluindo a diabetes mellitus 17 e hipercolesterolemia 18. No entanto, um ponto fraco importante do modelo tradicional HLI é que é um modelo de isquemia aguda 12,13, enquanto DAP humana provoca isquemia crónica, como resultado do desenvolvimento progressivo de lesões ateroscleróticas oclusivas nas artérias periféricas. Em uma tentativa de contornar essa fraqueza, Tang e seus colegas inicialmente desenvolvido um modelo de rato da oclusão arterial femoral gradual usando constrictors AMEROID 19, e do mesmo grupo posteriormente developed um rato modelo semelhante 20. Constrictors AMEROID foram descritos inicialmente na década de 1950 em um modelo canino de isquemia miocárdica crônica 21,22. Estes dispositivos têm uma manga de metal externo envolvendo uma camada interior de um material higroscópico, geralmente caseína, e quando colocado em torno de uma artéria que induzir a oclusão progressiva vaso à medida que absorvem humidade a partir dos tecidos circundantes. Na sua modificação do modelo, Yang et al. Constritores colocado em ambas as extremidades proximal e distai artéria femoral em locais análogos aos locais de ligação cirúrgica, e eles ligado aos ramos laterais da artéria femoral, como no modelo tradicional. Comparado a HLI aguda, isquemia induzida pelo constrictor ameróide resultou em menor expressão de genes inflamatórios e cisalhamento dependentes de tensão, menor recuperação do fluxo sanguíneo 4 - 5 semanas após a cirurgia, e menos necrose muscular 20. Com base nestas observações, considerou-se que a oclusão arterial gradual pode fornecer um modelo de PAD mais relevante para a doença humana. Notavelmente, no relatório original, foram examinados os efeitos de isquemia induzida por constritor ameróide apenas em ratinhos C57BL / 6 19, que são relativamente resistentes à necrose do músculo induzida por isquemia 15. Nós recentemente modificou o modelo de isquemia gradual mais e explorou seus efeitos no mais isquemia e suscetíveis BALB / c do mouse estirpe 23. Na primeira manifestação do modelo, colocado constrictors tanto no proximal e artéria femoral distal, mas deixou todos os ramos laterais intacta. Em uma segunda modificação, mais suave, colocamos um único constrictor apenas na artéria femoral proximal e novamente à esquerda todos os colaterais ramos da artéria intacta. Em ambas as modificações deste modelo, descobrimos que os ratos BALB / c, mas não camundongos C57BL / 6, exibido necrose muscular significativa, apesar de ter o fluxo sanguíneo semelhante e densidade vascular. Semelhante ao nosso estudo anterior 14, estes resultados demonstraram que muscular de membroso prejuízo não é apenas influenciado pelo fluxo sanguíneo, mas é em parte dependente de fundo genético. Além disso, descobrimos que o fluxo sanguíneo do membro caiu para seu ponto mais baixo dentro de 3 dias, assim, o modelo parece ser mais um dos &quot;subaguda&quot; em vez de isquemia gradual. Com base nestes estudos prévios, parece claro que um método único para a indução de isquemia dos membros posteriores pode não ser adequado em todos os casos. Por causa de uma variedade de condições (por exemplo, diferenças genéticas e a presença ou ausência de condições co-mórbidas) influenciam tanto a vascular e respostas específicas do músculo esquelético, os investigadores podem encontrar-se necessário modificar a cronicidade e / ou a gravidade de isquemia dos membros posteriores de melhor atender às suas finalidades. Além disso, as descrições anteriores do modelo normalmente faltava marcos anatômicos adequados para facilitar confiável reprodutibilidade inter-examinador da técnica. Neste trabalho, métodos para induzir isquemia dos membros posteriores aguda ou subaguda em ratossão descritos, e as estruturas anatómicas precisos são fornecidos.

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methods for acute and subacute murine hindlimb ischemia

doença arterial periférica (DAP) é uma das principais causas de morbilidade e mortalidade cardiovascular em países desenvolvidos, e modelos animais que reproduzem de forma fiável a doença humana são necessários para desenvolver novas terapias para esta doença. O modelo do rato de membro posterior isquemia tem sido amplamente utilizado para este fim, mas a prática padrão de indução de isquemia aguda por ligação da artéria femoral pode resultar em necrose do tecido substancial, comprometer a capacidade de os investigadores a estudar as respostas vasculares e dos tecidos do músculo esquelético para isquemia . Uma abordagem alternativa para a ligação da artéria femoral é a indução de oclusão da artéria femural gradual através da utilização de constritores AMEROID. Quando colocado em torno da artéria femoral na mesma ou em diferentes locais, como os locais de ligação da artéria femoral, estes dispositivos de oclusão da artéria mais de 1 - 3 dias, resultando em mais gradual, isquemia subaguda. Isto resulta em necrose do tecido muscular esquelético menos substancial, Which pode imitar mais de perto as respostas observadas nos PAD humano. Porque genéticos influências fundo resultados em ambos os modelos de isquemia aguda e subaguda, a consideração da estirpe de ratinhos em estudo é importante na escolha do melhor modelo. Este artigo descreve o procedimento adequado e colocação anatômica de ligaduras ou constrictors AMEROID na artéria femoral do rato para induzir subaguda ou isquemia dos membros posteriores aguda no mouse.

identificação adequada da vasculatura do mouse hindlimb é fundamental para garantir a reprodutibilidade das técnicas para induzir tanto subaguda e isquemia dos membros posteriores aguda, conforme descrito aqui. Além da variação inerente em estudos com animais, outros factores podem introduzir variabilidade na imagiologia de perfusão de Doppler a laser (LDPI), incluindo o tipo de anestesia, a posição do animal (comparada propenso supina), e a temperatura do corpo (ver <...

Watch this Scientific Journal Video about Methods for Acute and Subacute Murine Hindlimb Ischemia at JoVE.com

Methods for Acute and Subacute Murine Hindlimb Ischemia

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

Métodos para aguda e subaguda Murino dos membros posteriores Isquemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

The overall goal of this protocol is to model human peripheral artery disease by the placement of sutures or ameroid constrictors on the mouse femoral artery for the induction of acute or subacute hindlimb ischemia, respectively. Peripheral artery disease is an important clinical problem, and these techniques using animal models have been developed to better represent the human disease. The primary advantage of these techniques is that the severity and timing of the onset of limb ischemia can be controlled by the investigator.Demonstrating the procedure will be Mike Padgett, a technician from my laboratory. One to two days before the procedure, confirm that the mouse is fully sedated by lack of response to toe pinch, and apply eye ointment. Next, use a small electric trimmer to shave the hair from both hindlimbs.When all of the hair has been trimmed, apply pre-warmed hair removal cream. After one minute, gently wipe away the cream with a moistened gauze pad. Then place the mouse in an empty paper towel lined recovery cage until it is able to maintain sternal recumbency.On the day of the surgery, place the fully sedated mouse under a technic stereo dissection microscope in the supine position, and secure both legs with surgical tape. If a temperature controlled heating pad is used, attach the temperature probe, and secure the probe to the base of the surgical platform with surgical tape. Next, use three alternating povidone iodine and alcohol wipes to clean the surgical site, and cover the animal with a sterile surgical drape with a hole at the incision site.Using a scalpel, make an initial incision along the center of the medial thigh from the knee toward the abdomen. Then use fine scissors to lengthen the incision to approximately one centimeter. Using forceps, open the incision to expose the membrane covering the inguinal fat tissue.Then use closed forceps to pierce through the membrane, into the space between the fat tissue and the abdomen. Gently release the pressure on the forceps to separate the fat tissue from the abdominal muscles, exposing the neurovascular bundle. It is crucial that the major blood vessels and their branches are properly identified prior to the dissection of the neurovascular bundle.Variations in the location of the constrictors or the sites of ligation can significantly alter the severity of the ischemia, and lead to a substantial variability in the outcomes. Insert a retractor into the incision, and pull the abdominal tissue toward the animal's head to expose the proximal ameroid constrictor site, which is just proximal to the lateral circumflex femoral artery. Then insert one medial and one lateral retractor into the distal part of the incision to widen the surgical field, pulling the inguinal fat tissue toward the foot and away from the surgical site.The proximal and superficial caudal branches of the femoral artery should be easily observed. The lateral circumflex femoral artery is located about five millimeters proximal to the caudal branches, and may be obscured by membrane. Now gently slide the tip of one forceps under the membrane that binds the vessels together to insert 1/2 of the tip between the vein and the artery.Then closet he forceps and gently tear away the membrane. When all of the membrane has been removed, insert the tip of the closed forceps between the vein and the artery, and release the pressure on the forceps to create a gap between the vessels. For subacute limb ischemia, slide the tip of the forceps under the femoral artery to isolate the vessel from the neurovascular bundle.Use a second set of angled forceps to grip the edge of the constrictor, and guide the constrictor under the femoral artery. Then lay the artery into the constrictor slot. Next, position the distal constrictor on the femoral artery immediately proximal to where the artery bifurcates into the popliteal and saphenous arteries, and place the femoral artery into the constrictor slot.For acute limb ischemia, thread a 7-0 suture under the artery at the same position as the proximal constrictor, and ligate the artery. Then tie a second ligature about one millimeter distal to the first, and use spring scissors to transect the artery between the two ligatures. For the distal arterial transection, place the ligatures about one millimeter apart, just proximal to where the femoral artery bifurcates into the popliteal and saphenous arteries.Then close the incision with interrupted five-0 vicryl sutures, and move the mouse to a 37 degree Celsius heating pad beneath a laser doppler perfusion imager. Now turn on the imager, and launch the image capture software. Click the New Single Image icon to open the scanner setup window, and set the scan size to large, and the scan speed to four milliseconds per pixel.Change the X and Y values under the Scan Area units panel to the appropriate values, then click the Video and Distance tab, and arrange both of the hindlimbs within the scan area. Click Auto Distance, then click Next to open the Subject Details window, and enter the subject information. Click Next to move on to the scanning window.Then click the Start Measurement button to open the Confirm or Override Scan Distance dialog box, and click OK to begin the scanning process. When the scanning is complete, move the mouse to an empty recovery cage with monitoring until it is fully recovered. In addition to the variations inherent in animal studies, other factors can introduce variability within the laser doppler perfusion imaging, including the body temperature, and the position of the animal.The subacute hindlimb ischemia model is dependent on the quality of the ameroid constrictors, which can vary widely within a given batch. Laser doppler perfusion imaging should be performed immediately after the procedure while the animal is still anesthetized to demonstrate the effects of the surgery, and to establish a baseline post-operative level of perfusion. A complete loss of hindlimb perfusion should be observed after the induction of acute limb ischemia, whereas only a mild decrease in perfusion is typically observed after the ameroid constrictor placement in the subacute model.These procedures allow exploration of the effects of varying degrees of ischemia on vascular remodeling and skeletal muscle regeneration in the mouse as a model of human peripheral artery disease. Once mastered, these procedures can be completed in about 15 minutes if they are performed correctly. While attempting these surgeries, it is important to remember to continually monitor the body temperature and the adequacy of the anesthesia in the animal.By adjusting the placement of the ligatures or the constrictors, the severity of the ischemic injury can be modified, which can be an important variable depending on the animal's genetic background, or the presence of cardiovascular risk factors. After watching this video, you should have a good understanding of how to induce both acute and subacute hindlimb ischemia in the mouse, and to define the vascular anatomy to ensure a proper outcome and reproducibility of the procedure.

methods-for-acute-and-subacute-murine-hindlimb-ischemia

Research

JoVE Journal

Medicine

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

CO expirado 2 Medição em pacientes entubados com respiração espontânea ou do Departamento de Emergência

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

Medicina

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Um modelo fechado de tórax murino de isquemia do miocárdio e reperfusão

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Um método de pesquisa para detecção de isquemia miocárdica transitória em pacientes com síndrome coronariana aguda Utilizando Análise do segmento ST contínua

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

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 ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Lesão tecido isquêmico na Skinfold Câmara do mouse Dorsal: Um Modelo retalho de pele para investigar aguda isquemia persistente

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

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, ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Abrir traqueostomia ácido gástrico aspiração Modelo murino de lesão pulmonar aguda Resultados em Maximal aguda não-letal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

Combinado de microscopia Intravital e ultra-sonografia contraste aprimorado do membro posterior Mouse para estudar a vasodilatação induzida por insulina e perfusão muscular

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Avaliando a angiogênese terapêutica em um modelo murino de isquemia Retromembro

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

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, ...