Эта работа была поддержана грантом Пенсильвании Министерства здравоохранения. Доктор Мигель Гарсия Тоскано был докторской сотрудник в Университете Темпл, который финансируется Альфонсо Мартин Эскудеро Фонд в течение этого исследования. Мы хотим поблагодарить Илья Йорданов и Кевин Kotredes для изготовления видео.

1. Лигирование слепой кишки и прокол, как мышь Модель по правам Сепсис Для этой процедуры C57BL / 6 мышей (7-9 недель) используются. <ol><li> Анестезию мыши путем введения внутрибрюшинно раствор 1:1, кетамина (75mg/kg) и ксилазина (15мг/кг). В качестве примера можно вводить 30 мкл раствора в 1:01 мышь весом 20 граммов. Кроме того, мышь может быть анестезии ингаляционными изофлуран использования испарителя анестетиков. </li><li> Бритье брюшко мыши и дезинфекции области, первое применение бетадин решение следует вытирая с 70% спиртом (номер 1 на рисунке последовательности ниже). Хлоргексидин может быть использован как альтернативный антисептиком. Дополнительно: стерильный драпировка может быть использована для поддержания в чистоте. </li><li> В асептических условиях, практика 1 до 2 см срединной лапаротомии и подвергать слепой кишки с прилегающей кишечника (2 и 3). </li><li> Слепая кишка плотно лигировали с 6,0 шелковых шва (6-0 PROLENE, 8680G; Ethicon) на его базе ниже подвздошно-слепой кишки клапан, и перфорированные один или два раза с 19-иглы (4 и 5) на том же стороны слепой кишки. Пожалуйста, обратите внимание, что длина лигируют слепой кишки определяется как расстояние от дистального конца слепой кишки перевязки точка будет определять степень тяжести. Расстоянии&gt; 1 см производит высококачественных сепсисом при расстоянии ≤ 1 см производит средне-и низкосортных сепсиса. Также обратите внимание, что использование метода слепой кишки перфорация, показанный здесь, отличается от стандартного насквозь проколов техника (введение иглы через слепой кишки). Оба способа надежно производить тот же исход сепсиса. Однако один недостаток техники насквозь том, что она не способствует регулирования количества кала экструдированного, когда слепая кишка сжимается. </li><li> Слепая кишка затем мягко сжала чтобы выдавить небольшое количество кала от перфорации сайтов (6). Слепая кишка возвращается в брюшную полость и брюшины закрыта с 6,0 швов шелка. (7). </li><li> Кожа закрыта с 7 мм клипы Reflex (RS-9258, Roboz Хирургические инструменты) или Мишель раны клипов (7 мм, RS-9270) (8). </li><li> Resuscitate мышей путем введения подкожно 1 мл подогретого 0,9% солевого раствора с использованием 25G иглу. Эта жидкость мера реанимации будет побуждать гипердинамического фазы сепсиса. Это важный шаг описан далее в разделе обсуждения. </li><li> Дополнительно: Inject подкожно бупренорфин (0.05mg / кг массы тела) или трамадола (20mg/kg массы тела) для послеоперационного обезболивания. Имейте в виду, что эти опиатов может подавлять дыхание и передвижения, и эти эффекты могут быть ошибочно интерпретированы как признаки сепсиса. </li><li> Животные размещаются временно на грелку или же немедленно возвращены в клетку с воздействием инфракрасной лампой нагрева 150W, пока они не оправиться от наркоза. Время восстановления составляет от 30 мин до 1 часа. </li><li> Обеспечить свободный доступ к пище и воде (гидрогель), размещенные на дно клетки. </li><li> Мыши контролируются каждые 12 часов за выживание в течение одного-двух недель или эвтаназии в различные моменты времени для анализа различных параметров. </li></ol> В качестве контрольных для опытно-конструкторских, шам животных будут следовать лапаротомии технику без перевязки и пункции. От шести до 12 часов после хирургического мышей процедуры станут вялыми и развитие лихорадки, пилоэрекции, диарея, сбившись, и недомогание, и все признаки сепсиса. Мыши с очень тяжелым сепсисом едва ли могут двигаться до смерти и демонстрируют резкое снижение температуры тела. На данном этапе мышам должны быть умерщвлены, чтобы избежать длительной боли и страданий. <img src="/files/ftp_upload/2860/2860fig0.jpg" alt="Рис 0" /> 2. Наиболее распространенные анализируемых параметров Для оценки результатов процедуры, различные параметры могут быть проанализированы в органах, клеточных экстрактов или другими биологическими жидкостями. Образцы могут быть собраны в различные моменты времени от 3 часов до одной недели после хирургического вмешательства. <ol><li> Мыши выживания. </li><li> Цитокины и хемокинов в сыворотке крови, брюшной полости и органов экстрактов. <ol><li> Интерлейкина-6: Производство и выпущен моноциты, дендритные клетки, макрофаги, В-клетки, Т-лимфоциты, гранулоциты, тучные клетки и многие другие типы клеток. Он играет важную роль в фазу острой реакции и воспаление. </li><li> Фактор некроза опухоли-α: Pleiotrophic цитокина, который играет центральную роль в воспалении и апоптоза. Подготовлено моноциты, макрофаги, нейтрофилы, дендритные клетки и фибробласты. </li><li> nterleukin-1β: Продюсеры моноциты, НК-клетки, дендритные клетки, В-клетки и Т-клеток. Вызывает лихорадку и синтез белков острой фазы. </li><li> Интерлейкин 10: Способствует фагоцитарной поглощения и Th2 ответа, но подавляет antigан презентации и провоспалительных Th1 ответа. </li><li> Интерлейкин 10: Способствует фагоцитарной поглощения и Th2 ответа, но подавляет презентации антигена и провоспалительных Th1 ответа. </li><li> Моноцитарный хемотаксиса белка-1 (также известный как CCL2): Новобранцы моноциты, ячеек памяти T, и дендритные клетки к местам повреждения тканей и инфекции. </li><li> KC (CXCL1): Продюсер макрофаги и эндотелиальные клетки. Мышь KC является мощным аттрактанта нейтрофилов и активатор. </li><li> RANTES: моноцитов хемоаттрактант. Он может chemoattract нестимулированных CD4 + / CD45RO + Т-клеток памяти и стимулировало CD4 + и CD8 + Т-клеток с наивны и памяти фенотипы. </li><li> Интерферон-γ: выделяемый Th1 клеток, цитотоксических Т-лимфоцитов, дендритных клеток и естественных киллеров (НК) клетки. Увеличивает презентации антигена и литической активности в макрофагах и подавляет активность клеток Th2. Способствует адгезии и обязательным, необходимые для миграции лейкоцитов и способствует активности клеток NK. </li><li> Высокая мобильность группы В-1 белка: в моноцитов является ядерного фактора каппа-B активатора путем связывания с клеточным рецептором для продвижения гликирования конечных продуктов (RAGE), активизируя высвобождение провоспалительных медиаторов в поздней стадии сепсиса. </li></ol></li><li> Миелопероксидаза определения в органах в качестве меры нейтрофилов инфильтрации. Миелопероксидазы (МПО) является ферментом пероксидазой наиболее обильно присутствующие в нейтрофильных гранулоцитов. Это лизосомальных белков, хранящихся в азурофильных гранул нейтрофилов. МРО гема пигмент, который вызывает его в зеленый цвет выделений богат нейтрофилов, таких как гной и некоторые формы слизи. </li><li> Бактерии нагрузки в органах и крови, измерить как количество колониеобразующих единиц на миллилитр. </li></ol> 3. Представитель Результаты Процедура CLP изначально производится в штамм мыши C57BL / 6. Мы протестировали несколько параметров для модуляции тяжести сепсиса, изменяя длину труб и толщина иглы как показано на рисунках 1 и 2. Среди этих факторов, но длина перевязки, кажется, более эффективным, чем толщина иглы изменить процент выживания. Как показано на рисунке 1, увеличение длины перевязки более чем на 1 см провоцирует рост смертности на 100% по сравнению с мышами, имеющие перевязки ≤ 1см. Увеличение толщины иглы также снизился процент выживаемости с 100% (с помощью иглы 22G) до 55% (с помощью иглы 19G) с двумя проколами. Мы также проверили влияние на CLP C57BL / 6 и 129SvJ мышей для определения различных линий мышей дисплей аналогичных или различных восприимчивость к CLP-индуцированной сепсисом. Рисунок 3 показывает, что при тех же условиях, 129SvJ мышей были более восприимчивы к инфекции, чем C57BL / 6, свидетельствует увеличение смертности процентов. <img src="/files/ftp_upload/2860/2860fig1.jpg" alt="Рисунок 1" /> Рисунок 1. Влияние перевязки длина слепой кишки на животных выживание. CLP был выполнен в C57BL / 6 мышей с использованием двух различных длин слепой кишки труб. Это единственный параметр существенно влияет на выживаемость животных, так как все животные в группе с перевязкой площадь более 1 см умер менее чем за 3 дня по сравнению с группой животных, имеющих перевязки площадью 1 см прим. (П = 8). <img src="/files/ftp_upload/2860/2860fig2.jpg" alt="Рисунок 2" /> Рисунок 2. Влияние толщины иглы на выживание. CLP был выполнен в C57BL / 6 мышей с использованием двух различных размеров иглы 19G и 22G. Мыши с использованием перфорированной слепой кишки 19G иглу показали 55-60% выживаемости. В отличие от мышей с использованием перфорированных слепой кишки 22G иглу показали 100% выживаемости, хотя они испытывали типичные симптомы воспаления в течение первых 3-4 дней, которые исчезли после 4 дней (п = 8). <img src="/files/ftp_upload/2860/2860fig3.jpg" alt="Рисунок 3" /> Рисунок 3. Сравнение сепсис восприимчивости между двумя различными штаммами мыши. Модель CLP был выполнен в 129SvJ и C57BL / 6, на тех же условиях. В конце периода оценки процент выживания 129SvJ штамм был на 25% меньше, чем в C57BL / 6, напряжение, что указывает на более высокую восприимчивость к воспалению индуцированных полимикробная инфекции. (П = 6).

<ol>
	<li>Angus, D. C. <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+severe+sepsis+in+the+United+States:+analysis+of+incidence,+outcome,+and+associated+costs+of+care.">Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care.</a> Crit Care Med. 29, 1303-1310 (2001).</li><li>Riedemann, N. C., Guo, R. F., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigma+of+sepsis.">The enigma of sepsis.</a> J Clin Invest. 112, 460-467 (2003).</li><li>Eichacker, P. Q., Gerstenberger, E. P., Banks, S. M., Cui, X., Natanson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+of+acute+lung+injury+and+acute+respiratory+distress+syndrome+trials+testing+low+tidal+volumes.">Meta-analysis of acute lung injury and acute respiratory distress syndrome trials testing low tidal volumes.</a> Am J Respir Crit Care Med. 166, 1510-1514 (2002).</li><li>Buras, J. A., Holzmann, B., Sitkovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+sepsis:+setting+the+stage.">Animal models of sepsis: setting the stage.</a> Nat Rev Drug Discov. 4, 854-865 (2005).</li><li>Doi, K., Leelahavanichkul, A., Yuen, P. S., Star, 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=Animal+models+of+sepsis+and+sepsis-induced+kidney+injury.">Animal models of sepsis and sepsis-induced kidney injury.</a> J Clin Invest. 119, 2868-2878 (2009).</li><li>Godshall, C. J., Scott, M. J., Peyton, J. C., Gardner, S. A., Cheadle, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+susceptibility+during+murine+septic+peritonitis.">Genetic background determines susceptibility during murine septic peritonitis.</a> J Surg Res. 102, 45-49 (2002).</li><li>De Maio, A., Torres, M. B., Reeves, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+determinants+influencing+the+response+to+injury,+inflammation,+and+sepsis.">Genetic determinants influencing the response to injury, inflammation, and sepsis.</a> Shock. 23, 11-17 (2005).</li><li>Diodato, M. D., Knoferl, M. W., Schwacha, M. G., Bland, K. I., Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+the+inflammatory+response+and+survival+following+haemorrhage+and+subsequent+sepsis.">Gender differences in the inflammatory response and survival following haemorrhage and subsequent sepsis.</a> Cytokine. 14, 162-169 (2001).</li><li>Turnbull, I. R. <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+age+on+mortality+and+antibiotic+efficacy+in+cecal+ligation+and+puncture.">Effects of age on mortality and antibiotic efficacy in cecal ligation and puncture.</a> Shock. 19, 310-313 (2003).</li><li>Miyaji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethyl+pyruvate+decreases+sepsis-induced+acute+renal+failure+and+multiple+organ+damage+in+aged+mice.">Ethyl pyruvate decreases sepsis-induced acute renal failure and multiple organ damage in aged mice.</a> Kidney Int. 64, 1620-1631 (2003).</li><li>Torres, M. B., De Maio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+exaggerated+inflammatory+response+after+CLP+correlates+with+a+negative+outcome.">An exaggerated inflammatory response after CLP correlates with a negative outcome.</a> J Surg Res. 125, 88-93 (2005).</li><li>Doi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AP214,+an+analogue+of+alpha-melanocyte-stimulating+hormone,+ameliorates+sepsis-induced+acute+kidney+injury+and+mortality.">AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.</a> Kidney Int. 73, 1266-1274 (2008).</li><li>Wilson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+resuscitation+attenuates+early+cytokine+mRNA+expression+after+peritonitis.">Fluid resuscitation attenuates early cytokine mRNA expression after peritonitis.</a> J Trauma. 41, 622-627 (1996).</li><li>Zanotti-Cavazzoni, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+resuscitation+influences+cardiovascular+performance+and+mortality+in+a+murine+model+of+sepsis.">Fluid resuscitation influences cardiovascular performance and mortality in a murine model of sepsis.</a> Intensive Care Med. 35, 748-754 (2009).</li><li>Hugunin, K. M., Fry, C., Shuster, K., Nemzek, J. A. <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+tramadol+and+buprenorphine+on+select+immunologic+factors+in+a+cecal+ligation+and+puncture+model.">Effects of tramadol and buprenorphine on select immunologic factors in a cecal ligation and puncture model.</a> Shock. 34, 250-260 (2010).</li></ol>

Нет конфликта интересов объявлены.<br /Эксперименты&gt; Животные были проведены в соответствии с Национальным институтом здравоохранения Руководство по уходу и использованию лабораторных животных, и утверждается ухода за животными и использовать комитета при Университете Темпл.

Здесь мы подробно показать, как выполнить модель CLP у мышей и модулировать степени тяжести. По сравнению с другими моделями животных сепсиса, CLP могут быть выполнены в любой мыши штамма разного возраста и пола. Это относительно простой и недорогой хирургической операции....

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th><th> Комментарии </th></tr><tr><td> Кетамин </td><td> Ketaset </td><td> 0856-2013-01 </td><td></td></tr><tr><td> Ксилазин </td><td> AnaSed </td><td> NADA # 139-236 </td><td></td></tr><tr><td> 29G инсулина шприц </td><td> Exel </td><td> 26028 </td><td></td></tr><tr><td> Шелковая нить, 6-0 PROLENE </td><td> Ethicon </td><td> 8680G </td><td></td></tr><tr><td> 19G и 25G иглы </td><td> BD </td><td> 305186 </td><td></td></tr><tr><td> Бритва </td><td> Генеральный питания </td><td> Генеральный питания </td><td></td></tr><tr><td> Инфракрасные лампы Отопление </td><td> Генеральный питания </td><td> Генеральный питания </td><td></td></tr><tr><td> Мишель раны клипы 7мм </td><td> Roboz Хирургическое Instr </td><td> RS-9270 </td><td></td></tr><tr><td> Ухо Loop маски </td><td> Fisher Scientific </td><td> 19-130-4181 </td><td></td></tr><tr><td> Препарирование ножницы </td><td> Roboz Хирургическое Instr </td><td> RS-6702 </td><td></td></tr><tr><td> Бетадин раствор </td><td> VWR </td><td> 63410-992 </td><td></td></tr><tr><td> Хирургические щипцы </td><td> Roboz Хирургическое Instr </td><td> RS-5135 </td><td></td></tr><tr><td> 70% изопропиловый спирт площадку </td><td> Fisher Scientific </td><td> 22-031-350 </td><td></td></tr><tr><td> Бупренорфин </td><td> Бедфорд Labs </td><td> 55390-100-10 </td><td></td></tr><tr><td> Трамадол </td><td> Sigma-Aldrich </td><td> 42965 </td><td></td></tr></tbody></table>

cecal ligation puncture procedure

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the host. Currently, sepsis ranks among the top ten causes of mortality in the USA intensive care units 1. During sepsis there are two established haemodynamic phases that may overlap. The initial phase (hyperdynamic) is defined as a massive production of proinflammatory cytokines and reactive oxygen species by macrophages and neutrophils that affects vascular permeability (leading to hypotension), cardiac function and induces metabolic changes culminating in tissue necrosis and organ failure. Consequently, the most common cause of mortality is acute kidney injury. The second phase (hypodynamic) is an anti-inflammatory process involving altered monocyte antigen presentation, decreased lymphocyte proliferation and function and increased apoptosis. This state known as immunosuppression or immune depression sharply increases the risk of nocosomial infections and ultimately, death. The mechanisms of these pathophysiological processes are not well characterized. Because both phases of sepsis may cause irreversible and irreparable damage, it is essential to determine the immunological and physiological status of the patient. This is the main reason why many therapeutic drugs have failed. The same drug given at different stages of sepsis may be therapeutic or otherwise harmful or have no effect 2,3. To understand sepsis at various levels it is crucial to have a suitable and comprehensive animal model that reproduces the clinical course of the disease. It is important to characterize the pathophysiological mechanisms occurring during sepsis and control the model conditions for testing potential therapeutic agents. 
To study the etiology of human sepsis researchers have developed different animal models. The most widely used clinical model is cecal ligation and puncture (CLP). The CLP model consists of the perforation of the cecum allowing the release of fecal material into the peritoneal cavity to generate an exacerbated immune response induced by polymicrobial infection. This model fulfills the human condition that is clinically relevant. As in humans, mice that undergo CLP with fluid resuscitation show the first (early) hyperdynamic phase that in time progresses to the second (late) hypodynamic phase. In addition, the cytokine profile is similar to that seen in human sepsis where there is increased lymphocyte apoptosis (reviewed in 4,5). Due to the multiple and overlapping mechanisms involved in sepsis, researchers need a suitable sepsis model of controlled severity in order to obtain consistent and reproducible results.

Watch this Scientific Journal Video about Cecal Ligation Puncture Procedure at JoVE.com

Cecal Ligation Puncture Procedure

Мышиной модели слепой кишки перевязки и проколы, как ценным инструментом для изучения человеческих сепсиса.

Слепой кишки Процедура прокола Лигирование

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the ...

Research

JoVE Journal

Medicine

54.4K Views.  Temple University. Мышиной модели слепой кишки перевязки и проколы, как ценным инструментом для изучения человеческих сепсиса.

Video: Cecal Ligation Puncture Procedure

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Коронарных артерий и Лигирование Intramyocardial инъекций в мышиной модели миокарда

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Измененный Техника для лигирования коронарной артерии у мышей

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Желчного протока Лигирование у мышей Индукция воспалительного печени травматизма и фиброза обструктивной холестаза

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Крысы модель Связывание печени раздела и воротной вены перевязку процедура поэтапного гепатектомии (ALPPS)

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

Пункция индуцированной неоваскуляризации Iris как модель мыши Рубеоз Радужки

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Пункция подключичной вены как альтернативный метод сбора образцов крови крыс

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

Сбор крови через Субключичную Вену пункцию у мышей

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

Перевязка левой коронарной артерии: хирургическая мышиная модель инфаркта миокарда

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In ...

Tail Vein Puncture Without Anesthesia

Пункция хвостовой вены без анестезии  

To begin, secure the rat in a plastic restraining cone, enclose the large end of the cone around the base of the tail. Ensure that the animal is comfortable and breathing is unrestricted throughout the procedure. Dip the tail in warm water for about one minute to dilate the vein, and dry the tail with a paper towel.In the restrainer, position the animal facing down with the tail lying on a heating pad. To select the appropriate tail vein for sampling, rotate the entire animal to the right or left side. Use the terminal third of the tail for blood vessel puncturing since the vessels become more superficial in this zone.Wipe the tail with 70%ethanol wipes at the puncture site and place it on the edge of the heating pad, creating an angle in the terminal third of the tail. This brings the vein to the surface and creates more space for taking the sample. For blood sampling, move the plunger back and forth in the syringe several times to smoothen the withdrawal and then pull on the plunger to create negative pressure in the syringe.Secure the tail flat on the heating pad using the non-dominant index and middle finger. Place the middle finger proximally and the index finger distally with the puncture site between these two fingers. Slide the insulin syringe against the index finger with the eye of the needle pointing upward until it is inserted into the vein.This creates an angle of 35 degrees between the needle and the tail. Once the needle has entered the vein, blood will flow into the syringe. Slowly withdraw the plunger of the syringe at a steady rate to collect the desired volume.Remove the syringe from the tail and allow a blood drop to form on the puncture side of the tail. Aspirate this blood to collect a few more microliters of blood, in case of a non-sterile procedure. Apply pressure to the puncture site to stop the bleeding and wipe the area with a new alcohol wipe.After removing the rat from the cone, return it to its cage. The success rates with the tail vein puncture methods were compared with the success rate of the penile vein puncture method under anesthesia. Interestingly, under anesthesia, the tail vein became very unreliable and had a success rate of only 25%The success rates showed almost similar results for the tail vein puncture in conscious rats and the penile vein puncture under anesthesia.The blood sampling was performed in less than six minutes in the tail vein puncture without anesthesia, and the penile vein puncture method under anesthesia, whereas the tail vein puncture method under anesthesia took more than eight minutes due to multiple failures.