A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

Summary

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

Abstract

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera.  Despite relatively challenging techniques, this procedure can be performed with >90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

Introduction

The spleen is the largest secondary lymphoid organ in the body and is critical in the immune and hematopoietic systems. Its functions are primarily carried out by two morphologically distinct compartments, the red pulp and the white pulp¹. The red pulp is a three-dimensional meshwork of venous sinuses and splenic cords that consist of reticular fibers, reticular cells, and associated macrophages. This unique structure allows the red pulp to act as an effective blood filter that removes foreign materials and old or damaged erythrocytes. The white pulp includes follicles, marginal zone, and the periarteriolar lymphoid sheaths (PALS) and is an imp....

Protocol

All procedures and animal use in this study were performed according to protocols approved by the Northwestern University Internal Animal Care and Use Committee (IACUC). In this study, 8 to 10 week old male CD45.2 and CD45.1 mice (both on BALB/c background, from Jackson laboratory) were used as spleen donors and recipients, respectively, to create syngeneic spleen transplantation models. All animals were housed in the sterile environment in the animal facilities of Northwestern University. The eye lubricant was applied t.......

Discussion

Compelling evidence suggests that spleen-derived monocytes play an important role in sterile inflammatory processes such as atherosclerosis¹⁹, acute ischemic brain²⁰ or lung injury¹⁸, as well as myocardial I/R injury and remodeling^21,22,23. These reports highlight the under-recognition role of the spleen in many chronic diseases, of which cardiovascular dise.......

Materials

Name	Company	Catalog Number	Comments
Ketamine	Wyeth	206205-01
Xylazine	Lloyd Laboratories	139-236
Heparin solution	Abraxis Pharmaceutical Products	504031
Injection grade normal saline	Hospira Inc.	NDC 0409-4888-20
70% Ethanol	Pharmco Products Inc.	111000140
ThermoCare Small Animal ICU System	Thermocare, Inc.
Adson Forceps	Roboz Surgical Instruments	RS-5230
Derf Needle Holder	Roboz Surgical Instruments	RS-7822
Extra Fine Micro Dissecting Scissors	Roboz Surgical Instruments	RS-5881
Micro-clip	Roboz Surgical Instruments	RS-5420
7-0 silk	Braintree Scientific	SUT-S 103
11-0 nylon on 4-mm (3/8) needle	Sharpoint DR4	AK-2119
Ms CD45.2 antibody	BD Bioscience	553772
Ms CD45.1 antibody	BD Bioscience	553776
Ms CD11b antibody	BD Bioscience	557657
Ms B220 antibody	BD Bioscience	553089
Ms Ly6C antibody	eBioscience	48-5932-80
Ms Ly6G antibody	BD Bioscience	561236
Ms F4/80 antibody	BD Bioscience	565614
Ms CD11c antibody	BD Bioscience	558079
Ms CD3 antibody	eBioscience	48-0032-82
Ms CD4 antibody	BD Bioscience	552051
Ms CD8 antibody	BD Bioscience	563786
LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit	Thermo Fisher	L34955