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Abstract

Introduction

Protocol

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Acknowledgements

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Medicine

Mouse Models for Graft Arteriosclerosis

Published: May 14th, 2013

DOI:

10.3791/50290

1Department of Surgery, Yale University School of Medicine , 2Department of Pathology, Yale University School of Medicine

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-γ in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-γ followed by administration of mouse IFN-γ (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree 7. Early stages may cause eccentric and focal stenoses that are more obvious in arteries, thus more closely resembling stenoses seen in conventional atherosclerosis. The lumen loss of the graft vessels results from intimal expansion due to infiltration of host T cells and macrophages and especially to accumulation of extracellular matrix and smooth muscle-like cells originated from graft, host or both 5, 13, 19, that is ....

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Mouse allograft and syngeneic graft transplantation model

All animal studies were approved by the institutional animal care and use committees of Yale University. For allograft model, segments of thoracic aorta from male, 4-5 week old WT (C57BL/6J) or IFN-γR-KO mice were interposed into the abdominal aorta of female recipient, 8-12 week old WT using an end-to-end microsurgical anastomotic technique (see next for details). For syngeneic graft model, segments of thoracic aortae from male, 4-5.......

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Mouse allograft arteriosclerosis (GA) model: In this model, a male donor aorta is transplanted into female recipient so that the host induces alloreactive T cell-mediated alloimmune responses against a minor Y antigen (the male-specific minor histocompatibility antigen H-Y) expressed on the graft 12, and in turn T cell-produced IFN-γ drives VSMC proliferation 20 as observed in other allograft transplantation models 2, 6, 8-10, 14. A segment of thoracic aorta from a .......

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The described protocols are focused on mouse GA models. The procedures can be applied to other graft transplantation models. These models include humanized xenograft (i.e. human coronary artery segments interposed into the infra-renal aortae of immunodeficient mice), and acute rejection mouse GA model (i.e. a mouse aorta from one genetic strain into another genetic strain recipient). Our described mouse models are more to close human GA lesions. The first model involves interposition of a vessel segment.......

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This work was supported by NIH grants R01 HL109420 to WM and AHA 9320033N to LY.

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Name Company Catalog Number Comments
Name of the Reagent Company Catalogue Number Comments (optional)
C57BL/6J (H-2b) Jackson Laboratories (Bar Harbor, ME) 000664 Donor (5 weeks)
Recipient (8-12weeks)
Ketamine Hydrochloride Injection Hospira Inc. NDC 0409-2053 Storage Solution(50 mg/ml)
Working Solution(5 mg/ml)
Xylazine Sterile Solution Lloyd Inc. NADA# 139-236 Storage Solution(100 mg/ml)
Working Solution(1 mg/ml)
Ketoprofen Fort Dodge Animal Health NDC 0856-4396-01 Storage Solution(100 mg/ml)
Working Solution-oral
(0.027 mg/ml)
Heparin Sodium Sagent Pharmaceticals NDC 25021-400 Storage Solution(1000 U/ml)
Working Solution(100 U/ml)
Saline solution (Sterile 0.9% Sodium Chloride) CareFusion AL4109
0.9% Sodium Chloride Injection Hospira Inc. NDC 0409-4888-10 To prepare the anesthetic
Petrolatum Ophthalmic Ointment Dechra Veterinary Products NDC 17033-211-38
Iodine Prep Pads Triad Disposables, Inc. NDC 50730-3201-1
Alcohol Prep Pads McKesson Corp. NDC 68599-5805-1
Microscope Leica MZ95
Micro Scissors Roboz Surgical Instrument Co. RS-5693
Spring Scissors F.S.T 15009-08 To transect the aorta of donor or recipient
Extra Narrow Scissors F.S.T 14088-10
Needle Holder/Forceps MICRINS MI1542 To hold the needle
Fine Forceps F.S.T 11254-20
Forceps F.S.T 11251-35
Standard Pattern Forceps F.S.T 11000-12
Forceps F.S.T 13011-12
LANCASTER Eye Speculum Zepf Medical Instruments 42-1209-07
Micro Vascular Clip Roboz Surgical Instrument Co. RS-6472
Micro Clip Applying Forceps With Lock Roboz Surgical Instrument Co. RS-5440
Black Polyamide Monofilament Suture AROSurgical Instruments Corporation Cat #T4A10Q07 10-0 suture, Needle=70 microns
Black Monofilament Nylon Suture Syneture
(Covidien)
SN-1956 6-0 suture
Non-Woven Songes McKesson Corp. Reorder No. 94442000
1 ml Syringe BD REF 309659
3 ml Syringe BD REF 309657
10 ml Syringe BD REF 309604
18G 1 1/2, Hypodermic Needle BD REF 305196
25G 7/8, Hypodermic Needle BD REF 305124
27G 1/2, Hypodermic Needle BD REF 305109
30G 1/2, Hypodermic Needle BD REF 305106
Hearting Pad Sunbeam Z-1228-001
Trimmer Wahl 9854-500
Table 2. Specific reagents and equipment.

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