प्रतिगामी और सूक्ष्म गणना टोमोग्राफी इमेजिंग के लिए तैयारी के रूप में परफ्यूज़न माउस कोरोनरी vasculature का भरना
Summary
कोरोनरी वाहिकाओं के विज़ुअलाइज़ेशन हृदय रोगों के बारे में हमारी समझ को आगे बढ़ाने के लिए महत्वपूर्ण है. यहाँ हम एक radiopaque सिलिकॉन रबर (MICROFIL) के साथ सूक्ष्म गणना टोमोग्राफी इमेजिंग (μCT) के लिए तैयारी में murine कोरोनरी वाहिका संरचना, perfusing के लिए एक विधि का वर्णन.
Abstract
Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (μCT). μCT imaging can scan at resolutions <10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo μCT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by μCT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for μCT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network – arteries, capillaries, and veins alike.
Protocol
Discussion
हृदय ऊतक के एक बहुत ही उच्च चयापचय की मांग है, और इसलिए कोरोनरी vasculature के द्वारा दिया खून से पोषक तत्वों और ऑक्सीजन की लगातार आपूर्ति की आवश्यकता है. कोरोनरी वाहिकाओं, जो कोरोनरी पोत एक प्रकार का रोग और रुक…
Declarações
The authors have nothing to disclose.
Acknowledgements
हम प्रोटोकॉल, डॉ. माइकल सिमंस, डा. रात बिताने का स्थान Hauch के, और सामान्य चर्चा के लिए उनके प्रयोगशालाओं में दोनों के सदस्यों के प्रारंभिक परीक्षणों के लिए धन्यवाद डॉ. केली स्टीवंस.
इस काम एनआईएच HL087513 और HL094374 P01 अनुदान द्वारा समर्थन है.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| 1 ml syringes | Becton Dickinson | BD-309602 | |
| 1/2cc insulin syringes with permanently attached 29G ½’ needles | Becton Dickinson | BD-309306 | |
| 2″ x 2″ Gauze pads | Med101store.com | SKU 2208 | |
| 24G ¾” Angiocath IV catheter | Becton Dickinson | BD-381112 | |
| 26G ½”gauge needles | Becton Dickinson | BD-305111 | |
| Adenosine | Sigma | A9251 | 1g/L in PBS for Vasodilation Buffer (with Papaverine) |
| Angled Graefe Forceps | Fine Science Tools | 11052-10 | |
| Cotton-tipped applicators: 6″ non-sterile | Cardinal Health | C15055-006 | |
| Curved Surgical Scissors | Fine Science Tools | 14085-09 | |
| Dissecting stereoscope and light source | Nikon | NA | NA |
| Dissecting Tray, 11.5 x 7.5 inches | Cole-Parmer | YO-10915-12 | Filled with tar for pinning down the mouse |
| Fine Curved Forceps | Aesculap | FD281R | Need two |
| Heparin, 5000 U/ml stock | APP Pharmaceuticals LLC | NDC 63323-047-10 | 1:100 dilution in water |
| KCl | Fisher | P217 | Saturated solution in H2O |
| Ketamin (Ketaset), 100 mg/ml stock | Fort Dodge, Overland Park, KS, USA | NDC 0856-2013-01 | Mixed as 130 mg/kg body weight, with Xylazine in 0.9% saline |
| Microfil | Flow Tech | MV-122 (yellow). Other color options are also available. | Mix 1:1 by weight, with 10% by volume of curing agent. Prepare just before injection, and vortex to ensure it is well mixed |
| Non-sterile Suture: 6-0, braided silk | Harvard Apparatus | 723287 | |
| Papaverine | American Regent Inc. | NDC 0517-4010-01 | 4mg/L in PBS for Vasodilation Buffer (with Adenosine) |
| Paraformaldehyde | Sigma | P6148 | Prepared as 4% solution |
| Perfusion Apparatus | See figure 2 | ||
| Spring Scissors | Fine Science Tools | 15018-10 | |
| Xylazine (Anased), 20 mg/gl stock | Lloyd Labs | NADA #139-236 | Mixed as 8.8 mg/kg body weight, with Ketamin in 0.9% saline |
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