माउस रीढ़ की हड्डी दो photon माइक्रोस्कोपी का उपयोग गर्भनाल के vivo इमेजिंग में
Summary
एक न्यूनतम इनवेसिव के लिए माउस स्पाइनल कॉलम को स्थिर और दोहरावदार प्रदर्शन प्रोटोकॉल<em> Vivo में</em> रीढ़ की हड्डी दो photon माइक्रोस्कोपी का उपयोग इमेजिंग वर्णित है. यह विधि एक रीढ़ की स्थिरीकरण डिवाइस और एक चतनाशून्य करनेवाली औषधि आहार को जोड़ती है श्वसन प्रेरित आंदोलनों को कम करने और उत्पादन कच्चे इमेजिंग डेटा कि कोई संरेखण या अन्य पोस्ट प्रोसेसिंग की आवश्यकता है.
Abstract
In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.
Protocol
Discussion
विधि यहाँ वर्णित के लिए अनुमति देता है स्थिर और घनी आबादी दो photon माइक्रोस्कोपी का उपयोग करके anesthetized चूहों की रीढ़ की हड्डी में फ्लोरोसेंट सेलुलर संरचनाओं के vivo इमेजिंग में दोहराव है. स्थिरता हासिल एक क…
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम राष्ट्रीय मल्टीपल स्केलेरोसिस के द्वारा समर्थित किया गया था सोसायटी अनुदान / RG4595A1 टी के.ए. आंकड़े और फिल्मों अनुकूलित और / या Davalos एट अल., जम्मू Neurosci तरीके से reprinted डीडी और NIH / NINDS NS051470 अनुदान, NS052189 और NS066361. 2008 Mar 30, 169 (1) :1-7 2008 कॉपीराइट Elsevier से अनुमति के साथ.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Rhodamine B dextran | Invitrogen | D1841 | 70 kDa, diluted in ACSF (3% w/v) |
| Ketamine HCl | Bionichepharma | NDC No: 67457-001-10 | Injectable, 50mg/ml |
| Anased | Lloyd Labs | NADA No: 139-236 | Xylazine injectable, 20mg/ml |
| Acepromazine | Vedco | NADA No: 117-531 | Injectable,10mg/ml |
| Artificial tears ointment |
Phoenix pharmaceutical |
NDC No: 57319-760- 25 |
Lubricant |
| Betadine | Fisher | 19-061617 | |
| McPherson-Westcott Scissors |
World Precision Instruments |
555500S | Curved, blunt-tip scissors |
| Straight Forceps | World Precision Instruments |
555047FT | Toothed tip forceps |
| Small vessel cauterize | Fine Science Tools | 18000-00 | |
| Gelfoam | Pharmacia,Pfizer Inc. | Mixer Mill MM400 | |
| Compact spinal cord clamps |
Narishige | STS-A | |
| Head holding adaptor | Narishige | MA-6N | |
| Gelseal | Amersham Biosciences Corp. |
80-6421-43 | |
| Lactated Ringers | Baxter Healthcare | 2B8609 | |
| Buprenex | Reckit Benckiser Pharmaceuticals Inc. |
NDC No: 12496- 6757-1 |
Buprenorphine, injectable |
| Baytril | Bayer | NADA 140-913 | Enrofloxacin, antibacterial injectable 2.27% (20ml) |
| Heating pad – Large | Fine Science Tools | 21060-10 |
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