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

Summary

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.

Abstract

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.

Introduction

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 individual^2,3. Aberrant thymocyte selection of the αβTCR repertoire leads to autoimmune disease or immune imbalance⁴ that mainly derive from 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 interactions^3,5. The study of fixed tissues has rendered critical information regarding the patterns of expression for thymocyte migratory cues in defined thymic microenvironments^5,6, 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 medulla^{7,8,9,10,11,12,13}. 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 cultures^14,15, 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 grafts^19,20. 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' 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.

Protocol

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 phosphate-buffered saline (PBS, pH 7.4) and place it on ice. It will be used for rinsing and storing the excised thymi from donor mice prior to the transplantation.
4. Proceed to euthanize newborn donor mice by decapitation, in accordance with ethical guidelines.
5. Place the mouse on sterile absorbent paper towels in a dorsal upright position and spray, and later wipe, the mouse abdomen with 70% ethanol.
6. Expose the thoracic cavity by making a superficial V-shaped incision at the level of the lower abdomen and cut the skin with a pair of straight 10 cm dissecting scissors following a ventral midline to leave an opening of about 0.5-1 cm at the chest level. Fold the skin over each side of the chest to expose the thoracic cavity.
7. Make two deeper 0.5-1 cm lateral incisions through the diaphragm and ribcage with the same type of scissors to access the superior mediastinum in the anterior thoracic cavity. The thymus should appear as two pale lobes right above the heart.
8. Place a set of curved forceps underneath the thymus and pull vertically to extract the complete organ. Prevent the foldback of the ribcage with a pair of forceps. If needed, use fine forceps to carefully tear apart the connective tissue surrounding the organ without disrupting the capsule before extracting the organ.
   NOTE: This step is facilitated by using a dissecting scope.
9. Submerge the isolated thymus into cold sterile 1x PBS (pH 7.4) previously displayed into a 60 mm sterile dish, and cut the connective isthmus with a scalpel to separate the thymic lobes. Remove any debris from the dish without harming the capsule.To minimize the time thymi are exposed, trim thyme lobes right before implant.
   NOTE: Each isolated newborn thymus will typically render six segments of about 1 mm wide for a maximum of three host mice when receiving implants in both eyes.
10. Repeat Steps 1.4-1.9 for each donor mouse. To minimize the time thymi are exposed, isolate one thymus at a time.

2. Thymus Implantation into the Anterior Chamber of the Eye

1. Prior to the transplantation, tag and weigh each recipient mouse.
2. Use a dose between 1-2% of isofluorane vapors to anesthetize the recipient mouse. Ensure proper anesthetization by the absence of reflex following toe pinch before starting the surgical procedure.
3. Proceed with thymus segments transplant as follows:
   1. Place the mouse in a side lateral recumbent position so that one eye is facing up directly exposed to the lens of the dissecting scope.
   2. Excise one of the isolated thymic lobes lying in the cold 1x PBS (pH 7.4)-filled 60 mm dish into pieces with up to 1 mm width for implant. Follow a zig-zag pattern to ensure that each segment contains thymic cortex and medulla. Use vannas scissors for trimming according to guidelines provided in Figure 1A.
      NOTE: Trimming of the thymus should be done right before implant to optimize tissue engraftment.
   3. Starting in the base of the cornea corresponding to the surgical area, introduce the tip of a 40 mm G18 needle to make a small incision into external cornea layers so that the dissecting scissors tip can be introduced.
   4. Make a 5-10 mm flank incision directly around the base of the cornea using vannas scissors while holding the cornea opening firmly to prevent resealing. Use forceps with flat ends to avoid tissue damage.
   5. Grasp the cut corneal epithelium and expose the opening by holding the cornea with a pair of flat-ended forceps while pushing a thymic segment through the opening. Wet the eye with sterile 1x PBS (pH 7.4) or artificial tears as needed to prevent tissue desiccation until the manipulation is complete.
   6. Press softly on the eye surface to slide the introduced tissue segment to a lateral position with respect to the pupil to preserve eye´s function. Ensure that the graft lies at a location opposite to the eye opening to prevent posterior interference with imaging by hazing.
   7. With the aid of flat forceps, press firmly, for about 3-5 s, the two sides of the corneal opening against each other to promote self-sealing. The surgery does not require any stapling or sewing.
4. If the transplants are performed on both eyes, turn the mouse over the opposite lateral side to directly expose the second eye to the dissecting microscope lens and repeat Steps 2.3.2-2.3.7.
5. After the surgery is completed, return the mouse to an empty cage prewarmed with a heat lamp.
6. Make sure that each transplanted mouse regains complete mobility and consciousness before placing it into a cage shared with other animals. This usually takes place within 1 h after the surgery.
7. Upon transplant, treat the mice with pain killers as needed, and provide the mice with acetaminophen at a concentration of 1.6 mg/mL in the drinking water.
8. Repeat Steps 2.2-2.9 for each recipient mouse.
9. Monitor post-operative animal activity as well as the appearance of the implanted eyes to detect potential health complications.

3. Confocal Imaging of Implanted Thymi using 3D Single Photon Fluorescence Confocal Microscopy

1. Anesthetize the recipient mouse using a dose between 1-2% of isofluorane vapors. Ensure proper anesthetization by the absence of reflex following toe pinch before starting the imaging procedure
2. Place the mouse on a fixed stage microscope platform in a side lateral recumbent position so that one eye is facing up. A heat pad is placed on the microscope platform to ensure constant body temperature of mice during recordings.
   NOTE: See Supplementary Figure 1 for details.
3. Insert the head of the mouse into a stereotaxic headholder and adjust the knob to restrain the mouse head on a lateral side position allowing direct access of the microscope objective to the eye holding the thymus graft.
4. Place the mouse snout into a gas mask to keep the animal anesthetized throughout procedure. This allows the adjustment of isofluorane vapors as needed.
5. To facilitate the stabilization of the eye during recordings while avoiding the retraction of the eyelids, pull back the eyelids while holding the eye at the corneal margin with a pair of tweezers that have their tips covered by a polythene tube which are attached to a UST-2 solid universal joint. This arrangement permits a steady fixation of the head and eye and provides flexibility without disruption of blood flow in the eye, as previously described²².
   NOTE: Supplementary Figures 1B, C show the complete assembly.
6. Add a few drops of sterile saline or artificial tears as the immersion liquid between the cornea and the lens, before placing the microscope objective on the mouse eye.
   NOTE:Additional drops are dispensed on need throughout the procedure to keep the microscope path and prevent eye drying.
7. First use low magnification (5X) lens to locate the thymus in the microscope field. Then switch to higher resolution (10, 20 and 40X) water immersion dipping objectives with long working distance. Avoid LSM photodamage and bleaching of the thymic implant by applying minimal laser power and reduce scanning time as much as possible. This is achieved by using the resonant scanner of the microscope.
   NOTE: The confocal microscope we use is equipped with resonant scanning mirrors capable of gathering images at 25 frames/s. Other brands have their own microscopes with resonant scanners.
8. Select the acquisition mode using the microscope software and start the resonant scanner mode. Then choose the XYZT imaging mode and configure the acquisition settings as follows:
   1. Turn the Argon laser on and adjust the power to 30% for fluorescence excitation.
   2. Choose an excitation laser line and set the acousto-optical beam splitter (AOBS) control for different emission wavelengths. In addition, to detect backscatter and delineate tissue structure, use reflection detection simultaneously.
      NOTE: Upon selecting a set of colors for excitation (GFP and RFP), the AOBS is automatically programmed to direct these excitation lines onto the specimen and transmit the emission between them. For instance, for GFP and RFP, we use narrow reflection bands for excitation light around 488 nm and 561 nm, respectively. This leaves broad bands for the collection of emitted fluorescence photons, thus reducing the need for laser power and acquisition time. In reflection mode, the AOBS is used as a 50/50 beam splitter to image reflected light in any wavelength away from the ones used for fluorescence emission detection.
   3. Collect the emission at selected wavelength, then select a resolution of 512×512 pixels and start live imaging by pressing the Live button, adjusting the gain levels as needed (typical gain is around 600 V).
   4. Define the beginning and end of the z-stack by focusing on the top of the thymic implant and select Begin, then move to the last plane that can be focused in the implanted thymus and select End. Use a z-step size of 5 µm. The software will automatically calculate the number of confocal planes.
   5. Choose the time interval for acquisition of each z-stack (Typically 1.5 to 2 seconds) and select the option Acquire until stopped for continuous imaging.
9. Press the Start button to initialize.
10. Image the grafts repeatedly at different times from the same animal by repeating Steps 3.1-3.9.
    NOTE: If the animal holds grafts on both eyes, recordings from either eye can be taken by repeating Steps 3.2-3.9 after switching the upright position side of the mouse head.

Results

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 ...

Discussion

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 thymus^2,3, 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...

Materials

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