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 in....

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 transplant....

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