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

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.