فإن الكتاب أود أن أشكر مختبر الدكتور كارول كيم للتدريب microinjection، كلاريسا هنري لتقديم المشورة بشأن تسريع تطور الجنين واستخدام المعدات، وناثان لوسون للمساهمة fli1: EGFP الأسماك. ونحن نشكر أعضاء المختبر ويلر والحوائط شون لقراءة نقدية للمخطوطة. ونود أيضا أن نشكر مارك Nilan للرعاية الأسماك والمشورة، وريان Phennicie وجابور كريستين لتقديم المشورة الفنية في هذا المشروع. وقد تم تمويل هذا العمل من خلال مساعدة مالية البحوث MAFES إلى الإخوة ك، وهي هاتش MAFES منحة E08913-08، وجائزة NCRR المعاهد الوطنية للصحة P20RR016463 إلى يلر ر.

الإعلان عن أي تضارب في المصالح.

طريقة microinjection الزرد المقدمة هنا يختلف عن Gutzman وآخرون (34). في ذلك هنا علينا أن نبرهن عن طريق حقن الحويصلة الأذنية إلى البطين الدماغ المؤخر من اليرقات HPF 36-48. الأسلوب وصفنا يسمح للحقن متسق من 10-15 الخميرة إلى البطين الدماغ المؤخر مع انخفاض تلف الأنسجة. هذا البروت...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليق (اختياري) </td></tr><tr><td> التفريخ الدبابات </td><td> الموائل المائية </td><td> 2L </td><td></td></tr><tr><td> 1.7 مل من أنابيب </td><td> Axygen </td><td> MCT-175-C </td><td></td></tr><tr><td> لحظة المحيط </td><td> فيشر العلمية </td><td> S17957C </td><td></td></tr><tr><td> إضافي عميق أطباق بتري </td><td> فيشر العلمية </td><td> 08-757-11Z </td><td></td></tr><tr><td> معيار أطباق بتري </td><td> VWR العلمية </td><td> 25384-302 </td><td></td></tr><tr><td> نقل ماصات </td><td> فيشر العلمية </td><td> 13-711-7M </td><td></td></tr><tr><td> خلاصة الخميرة </td><td> VWR ScientiFIC </td><td> 90000-726 </td><td></td></tr><tr><td> الببتون مادة تنشأ عن البروتينات </td><td> VWR العلمية </td><td> 90000-264 </td><td></td></tr><tr><td> سكر العنب </td><td> فيشر العلمية </td><td> D16-1 </td><td></td></tr><tr><td> أجار </td><td> VWR العلمية </td><td> 90000-760 </td><td></td></tr><tr><td> يمكن التخلص منها عدادة الكريات </td><td> VWR العلمية </td><td> 82030-468 </td><td></td></tr><tr><td> التخزين المؤقت فوسفات المالحة </td><td> VWR العلمية </td><td> 12001-986 </td><td></td></tr><tr><td> دومون Dumoxel الملقط </td><td> VWR العلمية </td><td> 100501-806 </td><td></td></tr><tr><td> خشبية المسامير </td><td> VWR العلمية </td><td> 10805-018 </td><td></td></tr><tr><td> KimWipes </td><td> VWR العلمية </td><td> 300053-964 </td><td></td></tr><tr><td> انخفاض تذوبالاغاروز </td><td> VWR العلمية </td><td> 12001-722 </td><td></td></tr><tr><td> الاغاروز للأطباق حقن </td><td> VWR العلمية </td><td> 12002-102 </td><td></td></tr><tr><td> المشتعلة براون Micropipette بولير </td><td> سوتر الآلات </td><td> P-97 </td><td></td></tr><tr><td> قضبان الزجاج المجوف </td><td> سوتر الآلات </td><td> BF120-69-10 </td><td> لقضبان زجاج أملس عن طريق التسخين فوق الموقد بنسن </td></tr><tr><td> ماصة صندوق التخزين </td><td> سوتر الآلات </td><td> BX10 </td><td></td></tr><tr><td> MPPI-3 نظام حقن </td><td> الأجهزة العلمية التطبيقية </td><td> MPPI-3 </td><td></td></tr><tr><td> عودة وحدة الضغط </td><td> الأجهزة العلمية التطبيقية </td><td> حدة حراسة الحدود </td><td></td></tr><tr><td> Micropipette حامل عدة </td><td> تطبيق صحيفةإد الأجهزة العلمية </td><td> MPIP </td><td></td></tr><tr><td> قدم تبديل </td><td> الأجهزة العلمية التطبيقية </td><td> FSW </td><td></td></tr><tr><td> Micromanipulator </td><td> الأجهزة العلمية التطبيقية </td><td> MM33 </td><td></td></tr><tr><td> المغناطيسي قاعدة </td><td> الأجهزة العلمية التطبيقية </td><td> المغناطيسي قاعدة </td><td></td></tr><tr><td> Tricaine الميثان سلفونات </td><td> الغربي الكيميائية المؤتمر الوطني العراقي. </td><td> MS-222 </td><td></td></tr><tr><td> تشريح نطاق </td><td> أوليمبوس </td><td> SZ61 أعلى SZX-ILLB2-100 قاعدة </td><td></td></tr><tr><td> متحد البؤر المجهر </td><td> أوليمبوس </td><td> تاسعا-81-1000 FV مع نظام المسح الضوئي ليزر متحد البؤر </td><td></td></tr><tr><td> TC-7 أسطوانة الثقافة طبلة نسيج مع 14 بوصة عجلة أنبوب اختبار </td><td> نيو برونزويك العلمي </td><td> TC-7 </td><td></td></tr><tr><td> التصوير صحون </td><td> شركة ماتيك </td><td> P24G-1.0-10-F </td><td></td></tr><tr><td> نصائح ماصة للتحميل الإبر </td><td> إيبندورف </td><td> 930001007 </td><td></td></tr><tr><td> شبكات لوحة سكب </td><td> التكيف أدوات العلم </td><td> TU-1 </td><td></td></tr><tr><td> المرحلة الساخنة </td><td> Bioptechs المؤتمر الوطني العراقي. </td><td> دلتا T-5 </td><td></td></tr><tr><td> شقة أداة البسط </td><td> VWR العلمية </td><td> 82027-486 </td><td></td></tr><tr><td> البلاستيك المناخل </td><td> الأواني أون لاين Knutsford </td><td> 12 سم </td><td></td></tr><tr><td> Parafilm </td><td> VWR العلمية </td><td> 52858-000 </td><td></td></tr><tr><td> دوامة جيني </td><td> VWR العلمية </td><td> 14216-184 </td><td></td></tr><tr><td> 16 × 150 مم الثقافة أنابيب </td><td> VWR العلمية </td><td> 60825-435 </td><td></td></tr><tr><td> Nanodrop </td><td> الحرارية العلمية </td><td> ND 2000 </td><td></td></tr><tr><td> الفينول الأحمر </td><td> VWR العلمية </td><td> 97062-478 </td><td></td></tr><tr><td> حمض الهيدروكلوريك </td><td> VWR العلمية </td><td> 87003-216 </td><td></td></tr><tr><td> كلوريد الصوديوم </td><td> VWR العلمية </td><td> BDH4534-500GP </td><td></td></tr><tr><td> بوكل </td><td> VWR العلمية </td><td> BDH4532-500GP </td><td></td></tr><tr><td> MgSO 4 </td><td> VWR العلمية </td><td> BDH0246-500GP </td><td></td></tr><tr><td> كاليفورنيا (NO 3) 2 </td><td> VWR العلمية </td><td> BDH0226-500GP </td><td></td></tr><tr><td> HEPES </td><td> VWR العلمية </td><td> BDH4520-500GP </td><td></td></tص&gt; <tr><td> Morpholinos </td><td> GeneTools، ذ م م </td><td></td><td></td></tr></tbody></table>

non-invasive imaging of disseminated candidiasis in zebrafish larvae

المبيضات نشر الناجمة عن المبيضات البيض الممرض هي مشكلة هامة سريريا في المستشفى الأفراد ويترافق مع معدل وفيات 1 تعزى 30 إلى 40٪ 6. ويتم التحكم عادة المبيضات الأجهزة عن طريق المناعة الفطرية، والأفراد ذوي العيوب الوراثية في الخلايا المناعية الفطرية مكونات مثل NADPH أوكسيديز بلعمية هم اكثر عرضة لالمبيضات 7-9. ولا يعرف سوى القليل جدا عن القوى المحركة للC. وقد أنشأت المبيضة البيضاء التفاعل مع الخلايا المناعية الفطرية في الجسم الحي. واسعة في الدراسات المختبرية التي من خارج جيم المضيفة المبيضة البيضاء تنبت داخل الضامة، ويتم تدمير بسرعة من قبل العدلات 10-14. الدراسات في المختبر، على الرغم من المفيد، لا يمكن أن ألخص المجمع في بيئة الجسم الحي، والذي يتضمن الساعة التي تعتمد على ديناميات مستويات خلوى، التجهيزات المصفوفة خارج الخلية، والاتصالات بين الخلايا 10، 15-18 </sup&gt;. للبحث في مساهمة هذه العوامل في المضيف الممرض التفاعل، من المهم جدا للعثور على كائن نموذج لتصور هذه الجوانب من عدوى غير جراحية في المضيف سليمة حية. اليرقة الزرد توفر مجموعة الفقاريات وتنوعا فريدا لدراسة العدوى. عن ال 30 يوما الأولى من يرقات اسماك الزرد تنمية لديها دفاعات المناعة الفطرية فقط 2، 19-21، وتبسيط دراسة الأمراض مثل داء المبيضات نشر التي تعتمد اعتمادا كبيرا على الحصانة الفطرية. حجم صغير والشفافية من يرقات اسماك الزرد تمكين التصوير ديناميات العدوى على المستوى الخلوي لكل من المضيف والممرض. ويمكن استخدام اليرقات المعدلة وراثيا مع الاستشعاع خلايا المناعة الفطرية للتعرف على أنواع معينة الخلايا المشاركة في عدوى 22-24. ويمكن استخدام تعديل المضادة للإحساس [أليغنوكليوتيد] (Morpholinos) لاسقاط المكونات المناعية المختلفة مثل NADPH أوكسيديز بلعمية ودراسة التغيرات في الاستجابة لfungaل إصابة 5. بالإضافة إلى المزايا الأخلاقية والعملية لاستخدام أقل الفقاريات الصغيرة، ويرقات اسماك الزرد توفر إمكانية فريدة من نوعها لصورة معركة ضارية بين الممرض والمضيف على حد سواء وintravitally في اللون. وقد استخدم الزرد إلى عدوى نموذج لعدد من البكتيريا المسببة للأمراض البشرية، وقامت بدور فعال في تقدم كبير في فهمنا للعدوى المتفطرات 3 و 25. ومع ذلك، إلا في الآونة الأخيرة أن العوامل الممرضة أكبر من ذلك بكثير مثل الفطريات قد استخدمت لنقل العدوى يرقة 5، 23، 26، وحتى الآن لم يكن هناك وصفا تفصيليا للمنهجية البصرية عدوى. هنا نقدم تقنيات لدينا microinjection بطين الدماغ المؤخر من الزرد 25 متزمت، بما في ذلك التعديلات التي نبذلها لالبروتوكولات السابقة. النتائج التي توصلنا إليها باستخدام نموذج الزرد اليرقات للعدوى فطرية تختلف من الدراسات في المختبر، وتعزيز الحاجة لدراسة intera المضيف الممرضction في البيئة المعقدة للمضيف بدلا من نظام مبسط للطبق بيتري 5.

Watch this Scientific Journal Video about Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae at JoVE.com

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

والتطوير السريع، وصغر حجمها والشفافية من الزرد هي مزايا هائلة لدراسة سيطرة المناعة الفطرية للعدوى<sup&gt; 1-4</sup&gt;. هنا علينا أن نبرهن تقنيات لاصابة يرقات اسماك الزرد باستخدام العوامل المسببة للأمراض الفطرية<em&gt; المبيضات البيض</em&gt; من جانب microinjection، المنهجية المتبعة في الآونة الأخيرة لتوريط بلعمية النشاط أوكسيديز NADPH في السيطرة على إزدواج الشكل الفطرية<sup&gt; 5</sup&gt;.

غير الغازية التصوير من المبيضات المنتشر في يرقات اسماك الزرد

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

non-invasive-imaging-of-disseminated-candidiasis-in-zebrafish-larvae

Research

JoVE Journal

Immunology and Infection

14.6K Views.  University of Maine. والتطوير السريع، وصغر حجمها والشفافية من الزرد هي مزايا هائلة لدراسة سيطرة المناعة الفطرية للعدوى 1-4. هنا علينا أن نبرهن تقنيات لاصابة يرقات اسماك الزرد باستخدام العوامل المسببة للأمراض الفطرية المبيضات البيض من جانب microinjection، المنهجية المتبع...

Video: Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

غير الغازية تصوير الزاجل كريات بيضاء والهجرة في الجسم الحي</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

غير الغازية التصوير الضوئي من الأوعية الدموية اللمفية على فأرة الحاسوب

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

نموذج غير الغازية من ممرض للأعصاب<em&gt; الإشريكية القولونية</em&gt; العدوى في الأطفال حديثي الولادة الجرذ

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

التيموس كاملة في الجرذان الكبار مع غير الغازية التنبيب الرغامي

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

نمذجة الأغشية المخاطية المبيضات في اليرقات الزرد عن طريق الحقن Swimbladder

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

التصوير غير الغازية من الاستجابة المناعية الفطرية في نموذج الزرد يرقات<em&gt; العقدية iniae</em&gt; العدوى

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

والطريقة غير الغازية ومن الناحية الفنية غير كثيفة للتحريض وPhenotyping التجريبية البكتيرية والالتهاب الرئوي في الفئران

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

غير الغازية<em&gt; في فيفو</em&gt; الإسفار التصوير البصري من مب النشاط التهابات باستخدام عامل التصوير الفلورسنت أكتيفاتابل

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

وهناك طريقة غير الغازية لعزل والنمط الظاهري الخلايا من الملتحمة

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

الكشف عن استعمار الإشريكيّة القولونية Enterohemorrhagic موريني واستضافة بنظام الإضاءة الحيوية غير الغازية في فيفو

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...