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Abstract

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Protocol

Representative Results

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Acknowledgements

Materials

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Biology

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

Published: October 2nd, 2018

DOI:

10.3791/58236

1School of Medicine and Dentistry, Universidad Católica de Valencia San Vicente Mártir, 2Unidad Mixta CIPF-UCV, Centro de Investigación Príncipe Felipe, 3Department of Medicine, University of Miami

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes´ dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Intrathymic T-cell differentiation and T-cell subpopulation selection constitute key processes for the development and maintenance of cell-mediated immunity in vertebrates1. This process involves a complex sequence of tightly organized events including the recruitment of progenitors from bloodstream, cell proliferation and migration, differential expression of membrane proteins, and massive programmed cell death for subsets selection. The result is the release of mature T-cells reactive to an ample spectrum of foreign antigens while displaying minimized responses to self-peptides, which end-up colonizing the peripheral lymphoid organs of the in....

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The Institutional Animal Care and Use Committee (IACUC) of the University of Miami approved all the experiments according to IACUC guidelines.

1. Isolation and Trimming of Newborn Thymi

  1. Prepare all reagents and instruments by autoclaving or other methods, ensuring sterile conditions.
  2. To minimize contaminations, perform all surgical procedures under a laminar flow hood.
  3. Prior to euthanizing donor mice, fill a 60 mm sterile dish with sterile prechilled 1x phosphat.......

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Thymus from newborn mice were isolated from B6.Cg-Tg(CAG-DsRed*MST)1Nagy/Jas mice as described in this protocol (Steps 1.1-1.9). In these transgenic mice, the chicken beta actin promoter directs the expression of the red fluorescent protein variant DsRed. MST under the influence of the cytomegalovirus (CMV) immediate early enhancer facilitating the tracking of implants.

To prevent tissue rejection, isogenic individuals .......

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Due to the importance of the T-cell maturation process for individual immune competency4 and the presumed impact of precursor cell dynamics on mature T-cells produced by the thymus2,3, extensive efforts have been invested to develop alternatives to the classical fixed tissue snapshot approach.

Although tissue slices and other explants are clearly superior in reproducing tissue architecture than monolayers or agg.......

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This work was supported by NIH grants R56DK084321 (AC), R01DK084321 (AC), R01DK111538 (AC), R01DK113093 (AC), and R21ES025673 (AC), and by the BEST/2015/043 grant (Consellería de Educació, cultura i esport, Generalitat valenciana, Valencia, Spain) (EO). Authors thank the SENT team at the Universidad Católica de Valencia San Vicente Mártir, Valencia, Spain and Alberto Hernandez at Centro de Investigación Príncipe Felipe, Valencia, Spain for their help with video filming and editing.

....

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Name Company Catalog Number Comments
Isofluorane vaporizer w/isofluorane Kent Scientific Corp VetFlo-1215
Dissecting scope w/light source Zeiss Stemi 305
Fine dissection forceps WPI 500455
Medium dissection forceps WPI 501252
Curved tip fine dissection forceps WPI 15917
Vannas scissors WPI 503371
Dissecting scissors WPI 503243
Scalpel WPI 500353
40 mm 18G needles BD 304622
Disposable transfer pipette Thermofisher 201C
Heat pad and heat lamp Kent Scientific Corp Infrarred
Ethanol 70% VWR 83,813,360
60 mm sterile dish SIGMA CLS430166
Sterile 1x PBS pH(7,4) Thermofisher 10010023
Sterile wipes Kimberly-Clark LD004
Drugs for pain management Sigma-Aldrich A3035-1VL
Saline solution or Viscotears Novartis N/A
Stereomicroscope Leica MZ FLIII
Head-holding adapter Narishige SG-4N-S
Gas mask Narishige GM-4_S
Confocal microscope Leica TCS SP5 II
Laminar flow hood Telstar BIO IIA

  1. Boehm, T., Hess, I., Swann, J. B. Evolution of lymphoid tissues. Trends in Immunology. 33, 315-321 (2012).
  2. Takahama, Y. Journey through the thymus: stromal guides for T-cell development and selection. Nature Reviews Immunology. 6 (2), 127-135 (2006).
  3. Dzhagalov, I., Phee, H. How to find your way through the thymus: a practical guide for aspiring T cells. Cellular and Molecular Life Sciences. 69 (5), 663-682 (2012).
  4. James, K. D., Jenkinson, W. E. &. a. m. p. ;., Anderson, G. T-cell egress from the thymus: Should I stay or should I go?. Journal of Leukocyte Biology. , (2018).
  5. Savino, W., Mendes-Da-Cruz, D. A., Smaniotto, S., Silva-Monteiro, E., Villa-Verde, D. M. Molecular mechanisms governing thymocyte migration: combined role of chemokines and extracellular matrix. Journal of Leukocyte Biology. 75 (6), 951-961 (2004).
  6. Petrie, H. T., Zúñiga-Pflücker, J. C. Zoned out: functional mapping of stromal signaling microenvironments in the thymus. Annual Review of Immunology. 25, 649-679 (2007).
  7. Bousso, P., Bhakta, N. R., Lewis, R. S., Robey, E. Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science. 296, 1876-1880 (2002).
  8. Ladi, E., Herzmark, P., Robey, E. In situ imaging of the mouse thymus using 2-photon microscopy. Journal of Visualized Experiments. (11), e652 (2008).
  9. Bhakta, N. R., Oh, D. Y., Lewis, R. S. Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment. Nature Immunology. 6, 143-151 (2005).
  10. Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes. Immunity. 31, 986-998 (2009).
  11. Le Borgne, M., Ladi, E., Dzhagalov, I., Herzmark, P., Liao, Y. F., Chakraborty, A. K., et al. The impact of negative selection on thymocyte migration in the medulla. Nature Immunology. 10, 823-830 (2009).
  12. Sanos, S. L., Nowak, J., Fallet, M., Bajenoff, M. Stromal cell networks regulate thymocyte migration and dendritic cell behavior in the thymus. Journal of Immunology. 186, 2835-2841 (2011).
  13. Witt, C. M., Raychaudhuri, S., Schaefer, B., Chakraborty, A. K., Robey, E. A. Directed migration of positively selected thymocytes visualized in real time. PLoS Biology. 3 (6), e160 (2005).
  14. Ramsdell, F., Zúñiga-Pflücker, J. C., Takahama, Y. In vitro systems for the study of T cell development: fetal thymus organ culture and OP9-DL1 cell coculture. Current Protocols in Immunology. , (2006).
  15. White, A., Jenkinson, E., Anderson, G. Reaggregate thymus cultures. Journal of Visualized Experiments. (18), e905 (2008).
  16. Dunn, K. W., Sutton, T. A. Functional studies in living animals using multiphoton microscopy. ILAR Journal. 49, 66-77 (2008).
  17. Caetano, S. S., Teixeira, T., Tadokoro, C. E. Intravital imaging of the mouse thymus using 2-photon Microscopy. Journal of Visualized Experiments. (59), e3504 (2012).
  18. Li, J., Iwanami, N., Hoa, V. Q., Furutani-Seiki, M., Takahama, Y. Noninvasive intravital imaging of thymocyte dynamics in medaka. Journal of Immunology. 179 (3), 1605-1615 (2007).
  19. Adeghate, E. Host-graft circulation and vascular morphology in pancreatic tissue transplants in rats. Anatomical Record. 251, 448-459 (1998).
  20. Adeghate, E. Pancreatic tissue grafts are reinnervated by neuro-peptidergic and cholinergic nerves within five days of transplantation. Transplant Immunology. 10 (1), 73-80 (2002).
  21. Speier, S., Nyqvist, D., Köhler, M., Caicedo, A., Leibiger, I. B., Berggren, P. O. Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye. Nature Protocols. 3 (8), 1278-1286 (2008).
  22. Speier, S., et al. Noninvasive in vivo imaging of pancreatic islet cell biology. Nature Medicine. 14 (5), 574-578 (2008).
  23. Morillon, Y. M., Manzoor, F., Wang, B., Tisch, R. Isolation and transplantation of different aged murine thymic grafts. Journal of Visualized Experiments. 99 (99), (2015).
  24. Liu, L. L., Du, X. M., Wang, Z., Wu, B. J., Jin, M., Xin, B., et al. A simplified intrathymic injection technique for mice. Biotechnic & Histochemestry. 87 (2), 140-147 (2012).
  25. Manna, S., Bhandoola, A. Intrathymic Injection. Methods in Molecular Biology. 1323, 203-209 (2016).
  26. Abdulreda, M. H., et al. High-resolution, noninvasive longitudinal live imaging of immune responses. Proceedings of the National Academy of Sciences of the United States of America. 108 (31), 12863-12868 (2011).

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