Intravital تصوير الغدة الصعترية باستخدام الماوس 2 فوتون الميكروسكوب
Summary
We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of “niches” within the thymus where T cell development occurs.
Abstract
ثنائي الفوتون المجهري (TPM) على الحصول على الصور في مناطق عميقة داخل الأنسجة والأعضاء. في تركيبة مع تطوير أدوات جديدة المجسم والعمليات الجراحية ، TPM تصبح تقنية قوية لتحديد "منافذ" داخل الأجهزة الخلوية وثيقة "السلوك" في الحيوانات الحية. بينما intravital التصوير يوفر المعلومات التي تمثل أفضل السلوك الحقيقي الخلوية داخل الجهاز ، فإنه أكثر وشاقة وتتطلب من الناحية الفنية من حيث المعدات المطلوبة / الإجراءات البديلة السابقين من التصوير اكتساب الجسم الحي. وبالتالي ، فإننا تصف عملية جراحية ورواية "المجسم" حامل الجهاز الذي يتيح لنا متابعة تحركات Foxp3 + الخلايا داخل الغدة الصعترية.
Foxp3 هو المنظم الرئيسي لتوليد الخلايا التائية التنظيمية (Tregs). وعلاوة على ذلك ، يمكن تصنيف هذه الخلايا وفقا لأصلهم : أي. وتسمى الغدة الصعترية ، متباينة Tregs "طبيعيا Tregs" (nTregs) ، كما سpposed محيطيا لتحويلها Tregs (pTregs). على الرغم من أنه تم الإبلاغ عن كمية كبيرة من البحوث في الأدب المتعلق النمط الظاهري وعلم وظائف الأعضاء من هذه الخلايا T ، ولا يعرف سوى القليل جدا عن التفاعلات في الجسم الحي مع الخلايا الأخرى. قد يكون هذا النقص بسبب غياب التقنيات التي من شأنها أن تسمح مثل هذه الملاحظات. البروتوكول وصفها في هذه الورقة يقدم علاجا لهذه الحالة.
لدينا بروتوكول يتكون من استخدام الفئران عارية التي تفتقر إلى التوتة الذاتية منذ لديهم طفرة المنضبطة في تسلسل الحمض النووي الذي يهدد تمايز بعض الخلايا الظهارية ، بما في ذلك الخلايا الظهارية التوتة. وكانت الفئران عارية غاما المشع وتشكيل العظام مع الكوسا (BM) من الفئران GFP / GFP Foxp3 – KI. بعد شفائهم BM (6 أسابيع) ، تلقى كل حيوان التوتة الجنينية زرع الكلى داخل الكبسولة. بعد قبول الغدة الصعترية (6 أسابيع) ، تم تخدير الحيوانات ، والتي تحتوي على الكلىتعرض الغدة الصعترية المزروعة ، حامل ثابت في جهازنا ، وأبقى في ظل الظروف الفسيولوجية في الجسم الحي للتصوير بواسطة TPM. لقد تم استخدام هذا النهج لدراسة تأثير المخدرات في توليد الخلايا التائية التنظيمية.
Protocol
Discussion
In this paper we demonstrated the procedures for two-photon imaging of thymocytes inside a living animal. We also described some parameters that one should carefully control, such as the continuation of blood flow and the maintenance of organ temperature during the imaging procedures. Nonetheless, despite careful efforts to keep the organ stable, motion artifacts such as “organ drifting” can occur. Posterior image correction can be performed by the development of algorithms specifically designed for this purpose. Further image analysis could also be the source of new protocols development which seeks to minimize errors.
The thymus is the organ where all T cells are produced and, therefore, it is the organ where immunologists interested in understanding the generation of γδ, CD4, or CD8 T cells will focus their attention. Most studies concerning T cells are based upon differences in the numbers and/or stability of these cells after different in vitro/in vivo manipulations. However, only after the in vivo visualization we could observe the interaction between cells of the immune system involved in maintaining homeostasis3-7. Therefore, the in vivo observation of thymocytes is probably one of the most important missing information to better understand T cell biology. Intravital TPM provides a detailed picture of T cell movements and interactions and we demonstrate here how it can be used for detailed thymocyte studies. However, every technique has its limitations. While intravital imaging acquisition is the most accurate system for reflecting cells behavior inside the body, it is also true that explanted image acquisition of organs is less laborious and has been used to collect important information about the immune system8,9. Moreover, one cannot deny intravital imaging methods require surgery to expose tissues and blood vessels in anesthetized animals, which per se could cause an alteration in the whole organ physiology10. Nevertheless, there are non-invasively methods that abolish the artifacts caused by the surgical procedure11 and new methods are being developed that better prepare in advance the animals to be used12. Therefore, new surgical procedures and tolls will minimize or bypass actual limitations of intravital imaging acquisition and become more and more accessible to the scientific community.
We have demonstrated that the method we have described is feasible and it reports all in vivo systemic manipulations, such drug administration, that we have used. Thus, we suggest the use of this method together with ex vivo techniques already available in order to complement and strengthen further studies concerning thymocytes development.
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. David Olivieri for critical review of this manuscript, Dr. Nuno Moreno for the logistic help to build our animal holder and heating pads and Dr. Vijay K. Kuchroo for the kind donation of Foxp3-KIgfp/gfp mice. This work is supported by “Fundação para Ciência e Tecnologia” (FCT, Portugal), grant # PTDC/EBB-BIO/115514/2009.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Rhodamine B ishothiocyanate-Dextran | Sigma-Aldrich | R9379 | prepare stock at 20 mg/ml |
| Two-photon microscope | Prairie Technologies Inc. | Prairie Ultima X-Y | |
| Ti:Sapphire laser | Coherent, Inc. | Chameleon Ultra Family | |
| 20x/1.00 NA immersion objective | Olympus Inc. | XLUMPLFLN 20XW | |
| Holder (Filters/Dichroic) | Chroma Technology Corp. | 91018 BX2 (U-MF2) | |
| 525 nm/50 filter | Chroma Technology Corp. | ET525/50m | |
| 595 nm/50 filter | Chroma Technology Corp. | ET595/50m | |
| 565 nm dichroic | Chroma Technology Corp. | 565dcxr | |
| Imaris software | Bitplane AG Inc. | Imaris | |
| Volocity | PerkinElmer Inc. | Volocity | |
| ImageJ | NIH, USA | ImageJ |
Referencias
- Bettelli, E. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 441, 235-238 (2006).
- Ramsdell, F., Zúñiga-Pflücker, J. C., Takahama, Y., Coligan, J. E. In vitro systems for the study of T cell development: fetal thymus organ culture and OP9-DL1 cell coculture. Current protocols in immunology. , (2006).
- Tadokoro, C. E. Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. J. Exp. Med. 203, 505-511 (2006).
- Mempel, T. R., Henrickson, S. E., Von Andrian, U. H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature. 427, 154-159 (2004).
- Miller, M. J. Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes. J. Exp. Med. 200, 847-856 (2004).
- Shakhar, G. Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo. Nat. Immunol. 6, 707-714 (2005).
- Hugues, S. Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity. 5, 1235-1242 (2004).
- Tang, Q. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat. Immunol. 7, 83-92 (2006).
- Le Borgne, M. The impact of negative selection on thymocyte migration in the medulla. Nature immunology. 10, 823-830 (2009).
- Baez, S. An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy. Microvascular research. 5, 384-394 (1973).
- Wang, B., Zinselmeyer, B. H., McDole, J. R., Gieselman, P. A., Miller, M. J. Non-invasive Imaging of Leukocyte Homing and Migration in vivo. J. Vis. Exp. (46), e2062-e2062 (2010).
- Barretto, R. P. J. Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy. Nature medicine. 17, 223-228 (2011).
