A subscription to JoVE is required to view this content. Sign in or start your free trial.
* These authors contributed equally
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.
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.
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....
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.......
The entire procedure of mouse spleen transplant can be completed within 90 min by experienced microsurgeons. Our laboratory has performed over 100 spleen transplants in mice. The success rate is over 90%, as defined by the survival of both recipient mouse and the spleen graft to post-operative day (POD) 1 or POD 7 (our study endpoint). The survival of the spleen graft was confirmed by the macroscopic appearance and flow cytometry analysis of the splenocytes. Based on our experience, the f.......
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 remodeling21,22,23. These reports highlight the under-recognition role of the spleen in many chronic diseases, of which cardiovascular dise.......
Authors thank Northwestern University Comprehensive Transplant Center and the Feinberg School of Medicine Research Cores program for resource and funding support. Specifically, flow Cytometry and histology services were provided by the Northwestern University Flow Cytometry Core Facility and Mouse Histology and Phenotyping Laboratory, respectively, both of which are supported by NCI P30-CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. We thank Mr. Nate Esparza for proofreading this manuscript.
....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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved