Microcirculation के intravital इमेजिंग के लिए माउस Cremaster बलवान तैयारी
Summary
एक ऊतक तैयारी दृश्य और रहने वाले microcirculation के प्रयोगात्मक हेरफेर के लिए वर्णित है. Anesthetized नर चूहों में, पतली, उच्च vascularized cremaster मांसपेशियों intravital माइक्रोस्कोपी arterioles, capillaries और venules सहित microvascular नेटवर्क अध्ययन के लिए तैयार है. यह तैयारी चूहों और hamsters के लिए आसानी से अनुकूलित है.
Abstract
Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12.
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of calcium signaling in the arteriolar endothelium.
Protocol
Discussion
यहाँ हम vivo में microcirculation के अवलोकन के लिए माउस में खुला cremaster मांसपेशियों तैयारी का वर्णन . यह प्रक्रिया "खुला" cremaster पहले 4 चूहे में वर्णित तैयारी के बाद modeled है . अभ्यास के साथ पूरे शल्य चिकित्सा की प्रक्रिया कम से कम 1 घंटे में पूरा किया जा सकता है. इस तैयारी के बहुमुखी प्रतिभा प्रयोगात्मक अभिव्यक्ती की एक किस्म के लिए अनुमति देता है और आसानी से hamsters के रूप में अच्छी तरह के रूप में चूहों के लिए अनुकूलित, प्रयोगात्मक मॉडल की एक किस्म के लिए इसी तरह फैशन में अध्ययन किया जा सक्षम है. तैयारी पुरुष जानवरों के लिए सीमित है और माप ऊतक के केंद्र की ओर किया जाना चाहिए, 14 की मांसपेशी के किनारों के पास क्षतिग्रस्त क्षेत्रों से परहेज है. यह भी मान्यता है कि, जबकि दबाव और प्रवाह वितरण बदल रहे हैं जब cremaster मांसपेशियों की परस्पर वाहिकाओं 16 काट रहे हैं, microvessels संवेदनशील और प्रतिलिपि प्रस्तुत करने योग्य डेटा के संग्रह के लिए उपयुक्त रहते हैं होना चाहिए. cremaster मांसपेशियों में microcirculation visualizing प्राथमिक सीमा चूहों में विशेष रूप से, लेकिन यह भी hamsters में परिपक्व जानवरों और आकार में वृद्धि के रूप में संयोजी ऊतक के buildup है. इसके अलावा, के रूप में पशुओं वसा मिल, इसे और अधिक मुश्किल हो जाता है संज्ञाहरण नियंत्रण क्योंकि pentobarbital lipophilic है और वसा ऊतकों में अवशोषित किया जा सकता है. सबसे अच्छा तरीका है आगे बढ़ने रोगी जा रहा है जबकि यह सुनिश्चित करना कि जानवर ° शरीर का तापमान 37 ~ पर बनाए रखा है डिग्री सेल्सियस जबकि उजागर ऊतक लगातार PSS के साथ सिंचित है.
Disclosures
The authors have nothing to disclose.
Acknowledgements
'लेखक की प्रयोगशाला में अनुसंधान स्वास्थ्य R37-HL041026 अनुदान, R01-HL086483 और R01-HL056786 (एसएसएस) और F32 HL097463 और T32 AR048523 संयुक्त राज्य अमेरिका के सार्वजनिक स्वास्थ्य सेवा से (पंजाब) के राष्ट्रीय संस्थान द्वारा समर्थित है.
Materials
| Name of the reagent or device | Company | Catalogue number | Comments |
|---|---|---|---|
| Sodium Chloride | Fisher | S642-212 | |
| Potassium Chloride | Sigma | P9541 | |
| Magnesium Sulfate | Sigma | M2643 | |
| Calcium Chloride | Sigma | C1016 | |
| Sodium Bicarbonate | Fisher | S233 | |
| Nembutal Sodium Solution | Lundbeck | NDC 67386-501-55 | Also referred to as sodium pentobarbital |
| Temperature Controller | Warner Instruments | TC-344B | Alternate: adjustable 12V DC power supply |
| Series 20 platform heater kit RH-2 | Warner Instruments | 64-0274 | Requires custom-built aluminum plate |
| Mega-10 Camera | Stanford Photonics | XR/Mega-10 | |
| MVX10 | Olympus | ||
| MV PLAPO 2XC lens | Olympus | ||
| MVX10 hybrid stand | Leeds | LBX-Hybrid | |
| Piper Control Software | Stanford Photonics | Program for Mega-10 camera | |
| Stereo Microscope | Nikon | SMZ645 | |
| Waterproof Silicone Sealant (Clear) | General Electric | 47970-72643-LW5000 | Other clear silicone sealants also work |
| 60 x 15mm Petri Dish | Fisher | 08-757-13A | |
| Sylgard | Dow Corning | 184 | |
| Microdissection Scissors | Fine Science Tools | 15003-08 | |
| Dumont Forceps | Fine Science Tools | #5/45 | |
| GP Millipore Express PLUS Membrane | Millipore | SCGPT05RE | |
| Minutiens Insect Pins | Austerlitz | M size 0.15 mm | |
| Compact Pet Trimmer | Wahl Clipper Corp. | Model 9966 | Clean after each use |
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