We thank Katherine Clark for technical assistance. A.D.R.G. is funded in part by 5K01MH097957-03

واستخدمت الكبار (3-4 أشهر من العمر) ذكور الفئران على مختلطة C57BL / 6 الخلفية في هذا البروتوكول. تم الاحتفاظ الحيوانات على 12 ساعة ضوء / دورة الظلام، وسمح حرية الوصول إلى الغذاء والماء. أجريت جميع الإجراءات المتبعة في هذا البروتوكول وفقا لبروتوكولات وافقت عليها اللجنة دريكسل جامعة المؤسسي رعاية الحيوان واستخدام. 1. إعداد المنطقة الجراحية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تطهير الجدول الجراحية مع 70٪ من الإيثانول، ثم تغطية على مقاعد البدلاء الجراحية كامل مع منصات ماصة وترتيب الأدوات الجراحية المتاخمة لالتجسيمي. </li><li style=";text-align:right;direction:rtl"> نصب المعدات التجسيمي دون الذراع مناور. ترتيب سادة التدفئة على التجسيمي وتعيين إلى 37 درجة مئوية. تجنب الانهاك الحيوان عن طريق وضع قطعة صغيرة من منشفة ورقية أو وسادة الجراحية بين الحيوان وسادة التدفئة. </li><li style=";text-align:right;direction:rtl"> تعقيمها باستخدام مقص، وقطع قطعة صغيرة من جلفوم تعقيمها في طبق بتري معقمة تحتوي على محلول ملحي معقم بنسبة 0.9٪ حتى الجرميةدى للاستخدام. ملاحظة: الحفاظ على بيئة عمل معقمة باستخدام أدوات تعقيمها والمستلزمات الجراحية المعقمة. إعادة تعقيم الأدوات الجراحية أثناء إجراء أو بين الحيوانات عن طريق غمس في التعقيم حبة لمدة 10-15 ثانية، حسب الحاجة. الحفاظ على قفازات نظيفة في جميع أنحاء الداخلي عن طريق فرك اليدين مع الايثانول 70٪، حسب الحاجة، لتطهير. </li></ol> 2. الإستعداد ماوس لجراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة الماوس من قفص المنزل وتزن (ز). </li><li style=";text-align:right;direction:rtl"> مكان الماوس إلى الأيزوفلورين غرفة الاستقراء ووضع الأكسجين إلى 2 لتر / دقيقة والمرذاذ الأيزوفلورين إلى 5 للحث على متن طائرة من التخدير الجراحي، حوالي 3-5 دقائق. مراقبة التنفس تباطؤ والشلل. تأكد من أن الفأر هو تخدير كامل باستخدام منعكس قرصة أخمص قدميه. </li><li style=";text-align:right;direction:rtl"> عند مخدرا الماوس بشكل كامل، ووضع في إطار التجسيمي، تأمين الأنف في مخروط الأنف، وهو يعلق مع أنابيب إلى الأيزوفلورين. إدراج أشرطة الأذن في قناة الأذن وتشديد، وضمان رأس غير مستقر. </li><li style=";text-align:right;direction:rtl"> يحلق رأسه من الأذن إلى الأذن، ومن بين العينين إلى خلف الأذنين. </li><li style=";text-align:right;direction:rtl"> تعقيم الجلد مع مناديل بالتناوب من ايزوبروبيل وحل betadine اليود، 3 مرات لكل منهما. </li><li style=";text-align:right;direction:rtl"> تطبيق الدموع الاصطناعية لكلتا العينين لمنعهم من الجفاف أثناء إجراء العمليات الجراحية. </li></ol> 3. إجراء العمليات الجراحية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رصد عمق التخدير بواسطة معسر إصبع القدم أو الذيل. الفأر هو في الطائرة الجراحية المناسبة عندما لا يكون هناك استجابة، وتنفس بطيء وحتى. </li><li style=";text-align:right;direction:rtl"> جعل شق الجلد مجاور للسهمي من خلف العينين إلى ما يقرب من بين الأذنين في واحدة، والحركة حازمة واحد باستخدام رقم 11 شفرة المشرط. نقل الجلد جانبا ومقطع الجانب الأيمن مع مرقئ. </li><li style=";text-align:right;direction:rtl"> الجمجمة واضحة من الغشاء المغطي باستخدام الجانب مملة من مشرط رقم 11 و القطن يميل تطبيقها. اختياريا، ومسح الجمجمة مع القطن يميل قضيب انخفض بنسبة 0.9٪ في محلول ملحي. السماح ليجف تماما. </li><li style=";text-align:right;direction:rtl"> لناجي مسطرة صغيرة، بمناسبة الحافة الأمامية من حج القحف في 1 ملم الذيلية إلى الدرز الإكليلي، والحافة اليسرى من حج القحف في 1 مم الوحشي للخياطة السهمي (الشكل 1)، مع علامة دائمة. ثم وضع علامة الحق والحدود الذيلية من حج القحف في 4 ملم من الغرز السهمي والإكليلي، على التوالي (الشكل 1). </li><li style=";text-align:right;direction:rtl"> باستخدام 0.5 مم مثقاب، والبدء في جعل حج القحف عن طريق حفر ببطء بعد مخطط علامة دائمة. تأكد من عدم اختراق الجمجمة تماما. اضغط بلطف على قطعة معزولة من العظم الجداري مع رقم 5 ملقط، مواطن الضعف وتفسح المجال للضغوط. عندما العظام ضعيفة ضعيفة بما فيه الكفاية في جميع أنحاء محيط، وقطعة عظم جاهز للإزالة. ملاحظة: إذا كانت صعوبة الخبرات محقق إزالة العظام في قطعة واحدة، وهذا يشير إلى أن العظام لم ضعيفة بما فيه الكفاية أثناء الحفر. النظر في حفر الجمجمة كذلك في الحيوانات اللاحقة لfacilitaإزالة سهلة الشركة المصرية للاتصالات للقطعة عظم. </li><li style=";text-align:right;direction:rtl"> باستخدام حقنة 10 مل مزودة G 23 إبرة، وتطبيق كمية صغيرة من المياه المالحة 0.9٪ لامتصاص العظم معزولة ومنطقة حفر. </li><li style=";text-align:right;direction:rtl"> نعلق الذراع مناور لمعدات التجسيمي. إرفاق الجديدة رقم 11 شفرة مشرط لصاحب التحقيق مع الجانب حاد النصل تواجه rostrally. ملاحظة: على الرغم من أن الأنسجة منقاري والذيلية تجربة حواف حادة وحادة النصل، على التوالي، والأضرار الميكانيكية الناجمة عن الإصابة اختراق يماثل في جميع أنحاء مدى الآفة. نلاحظ عدم وجود فروق ملموسة في السمات الرئيسية لدباق رد الفعل بما في ذلك upregulation من التعبير GFAP أو الانتشار، بين أقسام منقاري والذيلية. </li><li style=";text-align:right;direction:rtl"> حفظ الذراع مناور للخروج من الطريق، ورفع بعناية العظام معزولة باستخدام ملقط 5/45 الزاوية. إدراج غيض من ملقط إلى جانب العظام معزولة ورفع، وذلك باستخدام النفوذ لسحب قبالة قطعة من العظام تركت وراءها في واحد فولل حركة. ملاحظة: يجب الحرص على عدم طعن الدماغ أو تعكير صفو الجافية تحت الجمجمة. </li><li style=";text-align:right;direction:rtl"> تأخذ قطعة صغيرة من الرغوة جل امتصاص غارقة ووضع على الدماغ المكشوف لمنعها من الجفاف وامتصاص أي دم التي قد تكون موجودة. </li><li style=";text-align:right;direction:rtl"> مرة واحدة الرغوة جل في مكان، تأرجح الذراع تتلاعب في مكانه وضبط نصل إلى مركز حج القحف على رغوة جل. إزالة الرغوة جل وخفض النصل حتى يلمس طرف الجافية دون ثقب الجافية. علامة ظهري / بطني الإحداثيات باستخدام مقياس رنيه على ذراع العمودي للالتجسيمي. </li><li style=";text-align:right;direction:rtl"> باستخدام الذراع مناور، وانخفاض ببطء شفرة بدقة 3 مم في الدماغ. ويتحقق ذلك باستخدام علامات نطاق رنيه على الذراع مناور. السماح نصل إلى البقاء في المكان لمدة 5-10 ثانية. تحريك الذراع التجسيمي مع مرفق شفرة منقاري إلى الذيلية ثلاث مرات مما يتيح للشفرة للوصول إلى حدود منقاري والذيلية من حج القحف قبل أن ينتقل إلى opposiالشركة المصرية للاتصالات نهاية. ملاحظة: لا يتم إزالة الجافية قبل إدخال شفرة. وعلى النقيض من الجافية الفئران، وهو ~ 80 ميكرون في سمك 7، الجافية الماوس هو أرق بكثير (فقط عدد قليل من طبقات الخلايا سميكة) و لا تنتج مقاومة لا يستهان بها لنصل مشرط خلال الإدراج. استخدام شفرة مشرط جديدة لكل الماوس للتأكد من أن كل حيوان يتلقى إصابة مماثلة. </li><li style=";text-align:right;direction:rtl"> رفع ببطء الذراع التجسيمي، وإزالة شفرة من الدماغ. بعد إزالة شفرة، ووضع على الفور قطعة أخرى من جلفوم على سطح الدماغ لامتصاص أي الدم الزائد أو السائل. </li><li style=";text-align:right;direction:rtl"> وفي الوقت نفسه، إزالة الذراع التجسيمي والتخلص من شفرة مشرط رقم 11. بمجرد أن توقف النزيف، وإزالة جلفوم. </li><li style=";text-align:right;direction:rtl"> إغلاق الجرح عن طريق خياطة الجلد مع خياطة غير قابل للامتصاص، مثل ethilon أو البرولين. يجب إزالة الغرز 9-10 أيام بعد الجراحة. </li><li style=";text-align:right;direction:rtl"> عودة الماوس إلى قفص وطنه، والسماح الماوس لاستعادة ببطء على اتسخينز وسادة ورصد أية علامات على الضيق. التعافي من التخدير التي يسببها isofluorane يحدث عادة خلال 2-5 دقيقة بعد إزالة من isofluorane. لا تترك الحيوان غير المراقب حتى استعادتها الإستلقاء القصية. </li><li style=";text-align:right;direction:rtl"> إدارة 0.5-1 مل من أفرز اللبن تحت الجلد حل قارع الأجراس لضمان الترطيب. </li></ol> 4. العناية ما بعد العمليات الجراحية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عن كثب مراقبة الحيوانات بعد العملية حتى الشفاء من التخدير قبل أن تعود إلى المستعمرة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> للحد من الألم وعدم الراحة بعد العملية، إدارة ،05-0،1 ملغم / كغم البوبرينورفين عن طريق الحقن الملكية الفكرية باتباع الإجراء فورا. </li><li style=";text-align:right;direction:rtl"> مراقبة الحيوانات لمدة 2-3 أيام بعد العملية عن علامات شديدة من الضيق مثل الحركات المحظورة، وعدم النظافة الشخصية، أو فقدان الوزن. الموت ببطء الحيوانات يدل أي من هذه العلامات الشدة وإزالته من الدراسة. </li></ol></li><li style=";text-align:right;direction:rtl"> لدراسة التشريح للأنسجة المصابين، الموت ببطء الحيوانات عن طريق ستاننضح intracardial دارد. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باختصار، تخدير الحيوانات مع جرعة زائدة من الكيتامين / زيلازين، ثم perfused لintracardially مع 15-20 مل من 0.9٪ كلوريد الصوديوم، أو حتى يتم مسح الكبد من الدم، تليها 60 مل من 4٪ لامتصاص العرق في ختام التجربة. </li></ol></li><li style=";text-align:right;direction:rtl"> تشريح أدمغة وبعد الإصلاح، لمدة 2-4 ساعة في 4٪ امتصاص العرق قبل ان ينتقل الى 30٪ محلول السكروز. أدمغة القسم على ناظم البرد في 40-60 ميكرومتر، والعملية من خلال إجراءات النسيجية أو المناعى القياسية، أو كما هو موضح في جارسيا 8،9. </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+Cells+Contribute+to+Myelin+Degeneration+and+Axonopathic+Changes+in+Mice+Overexpressing+Proteolipid+Protein+in+Oligodendrocytes.">Immune Cells Contribute to Myelin Degeneration and Axonopathic Changes in Mice Overexpressing Proteolipid Protein in Oligodendrocytes.</a> Journal of Neuroscience. 26 (31), 8206-8216 (2006).</li><li>Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+systemic+inflammation+to+chronic+neurodegeneration.">Contribution of systemic inflammation to chronic neurodegeneration.</a> Acta neuropathologica. 120 (3), 277-286 (2010).</li><li>McKeon, R. J., Jurynec, M. J., Buck, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chondroitin+sulfate+proteoglycans+neurocan+and+phosphacan+are+expressed+by+reactive+astrocytes+in+the+chronic+CNS+glial+scar.">The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar.</a> Journal of Neuroscience. 19 (24), 10778-10788 (1999).</li><li>Silver, J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+beyond+the+glial+scar.">Regeneration beyond the glial scar.</a> Nature reviews. Neuroscience. 5 (2), 146-156 (2004).</li><li>Zamanian, J. L., 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=Genomic+analysis+of+reactive+astrogliosis.">Genomic analysis of reactive astrogliosis.</a> J Neurosci. 32 (18), 6391-6410 (2012).</li><li>Buffo, 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=Origin+and+progeny+of+reactive+gliosis:+A+source+of+multipotent+cells+in+the+injured+brain.">Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (9), 3581-3586 (2008).</li><li>Sirko, 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=Reactive+glia+in+the+injured+brain+acquire+stem+cell+properties+in+response+to+sonic+hedgehog.+[corrected].">Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog. [corrected].</a> Cell stem cell. 12 (4), 426-439 (2013).</li></ol>

The authors have nothing to disclose.

فمن الأهمية بمكان أن الجمجمة أو الجافية الأساسية لا تلف أثناء الحفر. استخدام الضغط الخفيف أثناء الحفر لضمان الجمجمة لم يتم ثقب. بالإضافة إلى ذلك، يجب توخي الحذر في حين رفع قطعة الجمجمة لضمان الجافية لا انطلق مع العظم. إصابة الدما?...

A major challenge for developing successful therapies to treat the injured CNS is an incomplete understanding of the complex multicellular events that are triggered by the trauma. Reactive astrocytes are gaining increasing recognition as a promising target for novel therapies1. Though historically regarded as hostile to neural repair, reactive astrocytes are now recognized as critical components of a complex, multicellular neuroprotective response that includes attenuation of inflammatory processes and limiting secondary damage and neurodegeneration2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined, the cellular and molecular mechanisms regulating reactive gliosis, and the diverse array of downstream consequences remain poorly understood. Understanding the mechanisms that drive reactive gliosis, as well as the subsequent cellular and molecular events, is an important step towards developing strategies aimed at promoting the neuroprotective properties of reactive gliosis, while attenuating the detrimental effects. 
Here we demonstrate a method to induce severe reactive astrogliosis in the forebrain of adult mice using a stab injury. In contrast to other traumatic brain injury (TBI) models, such as controlled cortical impact (CCI) or fluid percussion injury (FPI), which require specialized equipment to produce an injury, the forebrain stab requires only a stereotaxic device to stabilize the head and a No. 11 scalpel blade. Thus the forebrain stab lesion model is more broadly accessible to a wide range of laboratories that do not have access to the specialized devices necessary for creating an FPI or CCI injury. The method described here enables investigators to reliably and reproducibly trigger a robust gliosis response to investigate subsequent cellular and molecular events. Once recovered from surgery, animals that have received a forebrain stab injury can survive for prolonged periods without the need for specialized care and can be returned to the colony for acute, intermediate, or chronic studies. Though less clinically translatable than FPI or CCI models of TBI, a forebrain lesion produced by a stab injury serves as a simple yet useful experimental model to investigate basic biological mechanisms underlying reactive gliosis and other neuropathological events following trauma to the CNS.

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triggering reactive gliosis in vivo by a forebrain stab injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

لأن الحيوانات تمر هذا الإجراء لا يتطلب تدرج فترات بقاء المتخصصة الرعاية اللاحقة للعمليات الجراحية، قصيرة أو طويلة الأجل بسهولة في الدراسة، وهذا يتوقف على الحاجة إلى تحقيق أمراض حادة أو مزمنة بعد الاصابة. الملامح الرئيسية لدباق رد الفعل، مثل upregulation من GFAP وتضخم في سو?...

Watch this Scientific Journal Video about Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury at JoVE.com

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

مما اثار رد الفعل دباق<em&gt; في فيفو</em&gt; من الدماغ الأمامي الطعنة الإصابات

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

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Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

10.4K Views.  Drexel University. This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods. 

Video: Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

و في الجسم الحي النموذجي القوارض من الإصابات الناجمة عن انكماش ، وغير الغازية ورصد الانتعاش

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

زرع الشريان السباتي الكفة لتحريك القص من التوتر في الفئران المستحثة تصلب الشرايين

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

في الجسم الحي 19 F التصوير بالرنين المغناطيسي لتتبع الخليوي

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

القياس عن بعد داخل الرحم لقياس ماوس مقلص الضغط<em&gt; في فيفو</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

الكشف عن القيامة في فيفو قبل بيوسينسور كاسباسيتراكير

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

نضوب خلايا الفأر من Xenografts ورم الإنسان يحسن بشكل ملحوظ تحليل المصب من الخلايا المستهدفة

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

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.

فتح القصبة الهوائية المعدة حمض الطموح فأري موديل الحاد الرئة إصابة النتائج في القصوى الحاد غير قاتل الرئة الإصابات

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

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

استنساخه الشرياني التعرية الإصابات وذلك Infrarenal البطن الأبهري لقط في نموذج الفئران

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

العلاج التجديدي بخلية سوبراتشورويدال أوتوجرافت في الجافة المرتبطة بالسن البقعي: تقرير أولى في فيفو

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

نموذج إصابة المورفولوجية في فيفو لدراسة نيوروريجينيريشن في الطرفية والجهاز العصبي المركزي

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...