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 (Conseller&#237;a de Educaci&#243;, cultura i esport, Generalitat valenciana, Valencia, Spain) (EO). Authors thank the SENT team at the Universidad Cat&#243;lica de Valencia San Vicente M&#225;rtir, Valencia, Spain and Alberto Hernandez at Centro de Investigaci&#243;n Pr&#237;ncipe Felipe, Valencia, Spain for their help with video filming and editing.

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

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+lymphoid+tissues.">Evolution of lymphoid tissues.</a> Trends in Immunology. 33, 315-321 (2012).</li><li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nature Reviews Immunology. 6 (2), 127-135 (2006).</li><li>Dzhagalov, I., Phee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+find+your+way+through+the+thymus:+a+practical+guide+for+aspiring+T+cells.">How to find your way through the thymus: a practical guide for aspiring T cells.</a> Cellular and Molecular Life Sciences. 69 (5), 663-682 (2012).</li><li>James, K. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+regulate+thymocyte+motility+during+positive+selection+in+the+three-dimensional+thymic+environment.">Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.</a> Nature Immunology. 6, 143-151 (2005).</li><li>Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+chemotaxis+and+substrate+restriction+to+segregation+of+immature+and+mature+thymocytes.">Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.</a> Immunity. 31, 986-998 (2009).</li><li>Le Borgne, M., Ladi, E., Dzhagalov, I., Herzmark, P., Liao, Y. F., Chakraborty, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+negative+selection+on+thymocyte+migration+in+the+medulla.">The impact of negative selection on thymocyte migration in the medulla.</a> Nature Immunology. 10, 823-830 (2009).</li><li>Sanos, S. L., Nowak, J., Fallet, M., Bajenoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell+networks+regulate+thymocyte+migration+and+dendritic+cell+behavior+in+the+thymus.">Stromal cell networks regulate thymocyte migration and dendritic cell behavior in the thymus.</a> Journal of Immunology. 186, 2835-2841 (2011).</li><li>Witt, C. M., Raychaudhuri, S., Schaefer, B., Chakraborty, A. K., Robey, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+migration+of+positively+selected+thymocytes+visualized+in+real+time.">Directed migration of positively selected thymocytes visualized in real time.</a> PLoS Biology. 3 (6), e160 (2005).</li><li>Ramsdell, F., Z&#250;&#241;iga-Pfl&#252;cker, J. C., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+systems+for+the+study+of+T+cell+development:+fetal+thymus+organ+culture+and+OP9-DL1+cell+coculture.">In vitro systems for the study of T cell development: fetal thymus organ culture and OP9-DL1 cell coculture.</a> Current Protocols in Immunology. , (2006).</li><li>White, A., Jenkinson, E., Anderson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaggregate+thymus+cultures.">Reaggregate thymus cultures.</a> Journal of Visualized Experiments. (18), e905 (2008).</li><li>Dunn, K. W., Sutton, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+studies+in+living+animals+using+multiphoton+microscopy.">Functional studies in living animals using multiphoton microscopy.</a> ILAR Journal. 49, 66-77 (2008).</li><li>Caetano, S. S., Teixeira, T., Tadokoro, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+the+mouse+thymus+using+2-photon+Microscopy.">Intravital imaging of the mouse thymus using 2-photon Microscopy.</a> Journal of Visualized Experiments. (59), e3504 (2012).</li><li>Li, J., Iwanami, N., Hoa, V. Q., Furutani-Seiki, M., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intravital+imaging+of+thymocyte+dynamics+in+medaka.">Noninvasive intravital imaging of thymocyte dynamics in medaka.</a> Journal of Immunology. 179 (3), 1605-1615 (2007).</li><li>Adeghate, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-graft+circulation+and+vascular+morphology+in+pancreatic+tissue+transplants+in+rats.">Host-graft circulation and vascular morphology in pancreatic tissue transplants in rats.</a> Anatomical Record. 251, 448-459 (1998).</li><li>Adeghate, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+tissue+grafts+are+reinnervated+by+neuro-peptidergic+and+cholinergic+nerves+within+five+days+of+transplantation.">Pancreatic tissue grafts are reinnervated by neuro-peptidergic and cholinergic nerves within five days of transplantation.</a> Transplant Immunology. 10 (1), 73-80 (2002).</li><li>Speier, S., Nyqvist, D., K&#246;hler, M., Caicedo, A., Leibiger, I. B., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+high-resolution+in+vivo+imaging+of+cell+biology+in+the+anterior+chamber+of+the+mouse+eye.">Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye.</a> Nature Protocols. 3 (8), 1278-1286 (2008).</li><li>Speier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+in+vivo+imaging+of+pancreatic+islet+cell+biology.">Noninvasive in vivo imaging of pancreatic islet cell biology.</a> Nature Medicine. 14 (5), 574-578 (2008).</li><li>Morillon, Y. M., Manzoor, F., Wang, B., Tisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transplantation+of+different+aged+murine+thymic+grafts.">Isolation and transplantation of different aged murine thymic grafts.</a> Journal of Visualized Experiments. 99 (99), (2015).</li><li>Liu, L. L., Du, X. M., Wang, Z., Wu, B. J., Jin, M., Xin, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+intrathymic+injection+technique+for+mice.">A simplified intrathymic injection technique for mice.</a> Biotechnic &amp; Histochemestry. 87 (2), 140-147 (2012).</li><li>Manna, S., Bhandoola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathymic+Injection.">Intrathymic Injection.</a> Methods in Molecular Biology. 1323, 203-209 (2016).</li><li>Abdulreda, M. 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The authors have nothing to disclose.

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...

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 individual2,3. Aberrant thymocyte selection of the &#945;&#946;TCR repertoire leads to autoimmune disease or immune imbalance4 that mainly derive from&#160;defects during the processes of negative or positive precursor selection, respectively.
Directional migration of thymocytes across the thymus is intrinsic to all stages of T-cell maturation and it is envisaged as a series of simultaneous, or sequential multiple stimuli, including chemokines, adhesive, and de-adhesive extracellular matrix (ECM) protein interactions3,5. The study of fixed tissues has rendered critical information regarding the patterns of expression for thymocyte migratory cues in defined thymic microenvironments5,6, while ex vivo studies has revealed two prevalent migratory behaviors of thymocytes in two histologically distinct areas of the organ: slow stochastic movements in the cortex and fast, confined motility in the medulla7,8,9,10,11,12,13. Increased migratory rates correlate with thymic positive selection13 and negative selection is associated with crawling behavior supporting the hypothesis that the kinetics of the journey through the thymus determines proper maturation of thymocytes. Despite their relevance, the topology of thymocyte-stromal cell interactions and the dynamics of thymocyte motility across organ microenvironments during T-cell maturation remain ill-defined.
Most ex vivo studies performed to date include fetal or reaggregate thymic organ cultures14,15, tissue slices or intact thymic lobe explants where thymocyte movements are visualized by two-photon laser scanning microscopy (TPLSM)8, an intravital imaging technique with a restricted maximum working distance and imaging depth of 1 mm in accordance with the tissue examined16. In contrast to the laborious thymic organ cultures which depend on extended incubation times to form 3D-structures, both, the thymic slice technique and the intact thymic lobe approach permit controlled introduction of particular subsets of pre-labeled thymocytes into a native tissue architecture environment. However, since blood flow is absent in these models, they are clearly limited for studying the recruitment process of thymus settling progenitors (TSPs) to the thymus parenchyma or the dynamics of thymic egression of mature T-cells.
In vivo models for the study of thymic T-cell maturation physiology in mice include the grafts of fragments or entire organ lobes placed either inside the kidney capsule17 or intradermally18. Although these options showed their utility to interrogate systemic functional engraftment of the tissue, the position of thymic grafts deep within the animal or covered by layers of opaque tissue restricts their use for in vivo examination of implants by TPLSM.
The anterior chamber of the eye provides an easily accessible space for direct monitoring of any grafted tissue by virtue of the transparency of corneal layers. Of advantage, the base of the chamber formed by the iris is rich in blood vessels and autonomic nerve endings, enabling rapid revascularization and reinnervation of the grafts19,20. Dr. Caicedo has successfully used this anatomical space for the maintenance and longitudinal study of pancreatic islets in the past21. Here, we show that this strategy not only constitutes a valid approach to study thymocytes&#39; dynamics within the native organ structure, but also uniquely permits to extend the in vivo longitudinal recordings to the study of progenitor recruitment and mature T-cell egression steps in mouse.

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

real time in vivo tracking of thymocytes in the anterior chamber of the eye by laser scanning microscopy

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&#180; 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.

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 ...

Watch this Scientific Journal Video about Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy at JoVE.com

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

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.

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

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 ...

This method can help answer key questions in immunology field about the mechanisms of autoimmunity, immunodeficiency, central tolerance, and thymus involution. The main advantage of this technique is that it permits in vivo longitudinal recording of progenitor recruitment and mature T-cell egression within vascularized and grafted mouse thymus segments. Though this method can provide insight into the development and function of the thymus, can also be applied to other tissues such as pancreatic islets or kidney glomeruli.To isolate the thymus, place the mouse on sterile absorbent paper towels in the dorsal upright position in a laminar flow hood and spray and wipe the mouse abdomen with 70%ethanol. Make a superficial V-shaped incision in the lower abdomen to expose the thoracic cavity and use a pair of straight 10-centimeter dissecting scissors to make a 0.5 to one-centimeter incision in the chest along the ventral midline. Fold the skin over each side of the chest to expose the thoracic cavity and make two additional deeper 0.5 to one-centimeter lateral incisions through the diaphragm and rib cage to access the superior mediastinum in the anterior thoracic cavity.The thymus should appear as two pale lobes right above the heart. Place curved forcep-tips under the thymus and pull vertically to extract the complete organ, preventing the fold-back of the ribcage with a pair of straight forceps. Then submerge the isolated thymus in a sterile 60-milliliter dish containing cold sterile PBS on ice and use a scalpel to cut the connective isthmus to separate the thymic lobes.Before transplantation, tag and weigh each recipient mouse and place the first animal in a side lateral recumbent position with one eye directly exposed to the lens of a dissecting microscope. Using Vannas scissors, excise one of the isolated thymic lobes up to one millimeter in width following a zigzag pattern to ensure that each segment contains some thymic cortex and medulla tissue. It is critical to trim the thymus right before the implantation as large pieces may not engraft properly.Next, starting at the base of the cornea of the surgical area, use the tip of a 40-millimeter 18-gauge needle to make a small incision into external cornea layers so that the tip of a pair of dissecting scissors can be introduced. Use the scissors to make a five to 10-millimeter flank incision directly around the base of the cornea while using flat-ended forceps to firmly hold the cornea open to prevent resealing. Grasping the cut corneal epithelium with the flat-ended forceps, maintain the opening in the cornea while inserting a thymic segment through the opening.It is important to insert the thymus piece quickly through the corneal opening to prevent spontaneous resealing before the tissue's introduced. Be careful to preserve tissue integrity at this step. Press softly on the eye surface to slide the introduced tissue segment to a lateral position with respect to the pupil to preserve the function of the eye.And use flat forceps to press both sides of the corneal opening firmly against each other for three to five seconds to promote self-sealing. If the transplant is to be performed on both eyes, turn the mouse onto the opposite lateral side for a second implantation as just demonstrated. When the surgery is complete, return the mouse to an empty cage pre-warmed with a heat lamp with monitoring until full recumbency.At the appropriate experimental time point, place an anesthetized recipient mouse on a fixed-stage microscope platform in a side lateral recumbent position on a heat pad with one eye facing up. And insert the head of the mouse into a stereotaxic head holder. Adjust the knob to restrain the mouse head in the lateral side position to allow direct access of the microscope objective to the eye holding the thymus graft and pull back the eyelids while holding the eye at the corneal margin with a pair of tweezer-tips covered by a polyethylene tube attached to a UST-2 solid universal joint.Add a few drops of sterile saline or artificial tears between the cornea and the lens before placing the 5X microscope objective on the mouse eye and locate the thymus in the microscope field. Then switch to a higher resolution water immersion dipping objective with a long working distance and select the acquisition mode in the microscope software. Start the resonance scanner mode and select the XYZT imaging mode.Turn the argon laser on and adjust the power to 30%for fluorescence excitation. Select an appropriate excitation laser line and set the acousto-optical beam splitter control for the appropriate emission wavelengths using reflection detection simultaneously to detect backscatter and to delineate tissue structure. Collect the emission at the selected wavelength and set a 512-by-512 pixel resolution.Then click live to start the imaging, adjusting the gain levels as necessary. Focus on the top of the thymic implant and select begin to define the beginning of the Z stack. Then focus on the last plane in the implanted thymus that can be visualized and select end to define the end of the Z stack using a Z-step size of five micrometers.Set the time interval for the acquisition of each Z stack for 1.5 to two seconds and select the acquire-until-stopped option for continuous imaging. Then click start to initialize the recording. Here, bright-field and fluorescence images of the same area of the thymus implant that may serve for a double macroscopic followup of tissue growth and involution and continuation studies are shown.The bright-field image also facilitates visualization of the vascularization of the implanted tissue, allowing the unique study of the dependence of thymus physiology on blood supply over time. This model allows the visualization of cell transmigration from the blood torrent into the implanted thymus, the tracking of the contacts between GFP-positive progenitor cells entering the thymic implant with RFP-labeled resident thymic epithelial and stromal cells during differentiation and selection processes, as well as the monitoring of the egressive immune cells from the thymus into peripheral blood vessels. While attempting this procedure, it's important to remember to minimize tissue exposure for optimal engraftment and also to limit incision size in the eye to permit unharmed fragment excision through the cornea while reducing fibrosis during healing.Following this procedure, other methods like localized control treatments and incision of other tissues into the contralateral eye can be performed so as to answer additional questions about immune dysfunctions linked to infections, involution determinants, and transplantation tolerance mechanisms. After its development, this technique paved the way for researchers in the field of immunology to explore T-cell development, including progenitor recruitment, thymus-eye dynamics, and mature T-cell egression steps in vivo.

real-time-vivo-tracking-thymocytes-anterior-chamber-eye-laser

Research

JoVE Journal

Biology

6.7K visualizações.  Universidad Católica de Valencia San Vicente Mártir. Este método pode ajudar a responder perguntas-chave no campo da imunologia sobre os mecanismos de autoimunidade, imunodeficiência, tolerância central e involução de timo. A principal vantagem dessa técnica é que permite o registro longitudinal in vivo do recrutamento progenitor e a egressão madura de células T nos segmentos de timo de camundongos vascularizados e enxertados. Embora este método possa fornecer uma visão sobre o desenvolvimento e a função do timo, também pode ser a...