Ischemia-reperfusion की चोट में ल्युकोसैट - endothelial सहभागिता के वास्तविक समय डिजिटल चूहा cremaster स्नायु की इमेजिंग (IRI)
Summary
डिजिटल cremasteric microcirculation में postcapillary venules intravital epifluorescence माइक्रोस्कोपी एक सुविधाजनक तरीका है लिए ल्युकोसैट – endothelial बातचीत में अंतर्दृष्टि लाभ<em> Vivo में</em> धारीदार मांसपेशी ऊतक की ischemia-reperfusion की चोट (IRI). हम यहाँ एक विस्तृत प्रोटोकॉल प्रदान करने के लिए सुरक्षित तकनीक का प्रदर्शन और उसके आवेदन और सीमाओं पर चर्चा.
Abstract
Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.
Protocol
Discussion
ल्युकोसैट endothelial बातचीत, प्रतिक्रियाशील ऑक्सीजन प्रजातियों के उत्पादन और पूरक व्यवस्था के सक्रियण, IRI प्रेरित ऊतकों में शिथिलता के प्रमुख विशेषताएं हैं 26 प्रभावित ऊतकों की microcirculation भड़काऊ शुरुआत के ल…
Declarações
The authors have nothing to disclose.
Acknowledgements
इस काम र Eisenhardt "ड्यूश Forschungsgemeinschaft (866/1-1 ईआइ) के एक अनुदान द्वारा समर्थित किया गया.
Materials
| Name of the equipment: | Company: | Catalogue No.: | Comments: |
| Forene 100% (V/V) | Abbot | B506 | API isoflurane |
| Terylene Suture | Serag Weissner | OC108000 | |
| Portex Fine Bore Polythene Tubing | Smiths Medical | 800/100/100 | 0.28 mm inner Diameter |
| 0,9% saline solution | Fresinus Kabi | 808771 | |
| Change-A-tip deluxe cautery kit | Bovie Medical | DEL1 | |
| Abbocath -T 14G | Venisystems | G713 – A01 | used as lens tube |
| Servo Ventilator 900C | Maquet | used as animal ventialtor | |
| Logical pressure transducer | Smiths Medical | MX1960 | |
| Sirecust 404 Monitor | Siemens | ||
| ABL 700 Benchtop Analyzer | Radiometer | for blood gas measurement | |
| Heating pad | Effenberger | 8319 | |
| Aluminum stage | Alfun | AW7022 | |
| Surgical microscope OPMI 6-SDFC | Carl Zeiss | ||
| Microsurgical instruments lab set | S&T | 767 | |
| Biemer vessel clip | Diener | 64.562 | |
| Applying forceps | Diener | 64.568 | for Biemer vessel clip |
| Rhodamine 6G | Sigma-Aldrich | R4127 | |
| Vaseline white DAB | Winthrop | 2726853 | |
| Cover glasses | 32×32 mm | ||
| Intravital setup | |||
| Zeis Axio Scope A-1 MAT | Carl Zeis | 490036 | epifluorescence microscope |
| 470 nm LED | Carl Zeis | 423052 | fluorescence light source |
| Colibri 2 System | Carl Zeis | 423052 | |
| W Plan-Apochromat 20x/1,0 DIC | Carl Zeis | 421452 | water immersion objective |
| AxioCam MRm Rev. 3 FireWire | Carl Zeis | 426509 | high resolution digital camera |
| Axio vision LE software | Carl Zeis | 410130 | use for offline analysis |
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