Establishment and Analysis of Acute Antibody-Mediated Rejection in Murine Cardiac Transplantation

Summary

This study established a practical mouse model of acute antibody-mediated rejection after cardiac transplantation, characterized by robust DSA production, typical pathological changes, and moderate allograft survival.

Abstract

A major contributor to allograft failure in cardiac transplant recipients is antibody-mediated rejection (AMR). The mouse AMR model, therefore, serves as a vital tool for deciphering its underlying mechanisms and fostering the development of innovative treatments. In this study, the recipient mice were divided into three groups: non-sensitized (NS), pre-sensitized (PS), and pre-sensitized with cyclosporine A treatment (PS + CsA). The NS group, which exhibited a mean allograft survival of 6.8 ± 0.7 days, showed no rise in serum DSA levels and negative allograft C4d staining within four days post-cardiac transplantation (CT), suggesting a pathology dominated by acute cellular rejection. This study established an acute AMR model by pre-sensitizing recipients with skin transplantation (ST) one week before CT, thereby pre-activating the immune system. This approach successfully induced a severe AMR phenotype, as evidenced by a short allograft survival of 2.8 ± 0.4 days, a significant rise in DSA-IgG levels post-ST and post-CT, and early pathological hallmarks of vasculitis and extensive C4d deposition within 12 h of CT. Nevertheless, the extreme severity of this model constrains its broader application. To minimize the concurrent T cell activation induced by ST and establish a more specific acute AMR model, this study administered cyclosporine A. Consequently, the PS + CsA group exhibited an allograft survival time of 5.2 ± 0.4 days. Serum DSA-IgG levels were significantly elevated by day 7 post-ST and remained high within five days post-CT. Pathological assessment on day 2 post-CT confirmed significant vasculitis and C4d deposition, findings which collectively meet the diagnostic criteria for moderately severe acute AMR. In conclusion, this study established a highly practical and translatable mouse model of acute AMR following CT, defined by robust DSA production, characteristic pathological changes, and moderate allograft survival.

Introduction

Cardiac transplantation (CT) remains the gold-standard therapy for end-stage heart disease¹. Although median post-transplant survival now exceeds 13 years, long-term graft failure remains inevitable². Antibody-mediated rejection (AMR) is a major contributor to late graft loss after CT³. Studies have shown that the incidence of graft loss due to AMR exceeds that caused by T cell-mediated rejection, and the risk of graft failure in late-onset AMR is approximately twice that of early AMR^4,5. Therefore, early identification and timely interven....

Protocol

All animal experiments were conducted in strict accordance with Guangdong experimental animal management regulations, the Declaration of Helsinki, and the 3Rs principles. The experimental protocol was approved by the Committee of Guangzhou Jennio Biotech Co., Ltd. (approval number JENNIO-IACUC-2024-A027). Male Balb/c and C57BL/6 mice (6-8 weeks old, weighing 20-25 g), all specific pathogen-free (SPF) grade, were obtained from a commercial source. The reagents and equipment used in this study are detailed in the Table of Materials.

1. Animal preparation

1. House the SPF grade mouse in the appropriat....

Results

Cardiac allograft survival time
Upon successful establishment of the mouse model, graft survival times across groups were assessed using Kaplan-Meier analysis. As anticipated, mice in the PS group exhibited significantly shorter mean survival of cardiac allografts compared to those in the NS group (2.8 ± 0.4 days vs. 6.8 ± 0.7 days, P < 0.01), attributable to acute AMR. To facilitate future evaluation of therapeutic strategies for acute AMR, CsA was administered to prolong allograft survival and w.......

Discussion

Currently, treatments for late-stage AMR remain ineffective, and reliable methods for its early diagnosis are lacking^22,23,24,25. To address this gap, this study established an acute AMR mouse model to facilitate mechanistic investigation. Recipients were pre-sensitized with ST and administered CsA to suppress concomitant aTCMR. Pathological analysis showed that allografts in the PS+CsA group d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-C4d Monoclonal Antibody	Hycult Biotech (Netherlands)	HP8034	For immunohistochemistry (detecting complement deposition)
Complete set of microsurgical instruments	Guangzhou Qihua Medical Equipment Co., Ltd.		Used for performing mouse cardiac transplantation surgery
Continuous zoom stereoscopic surgical microscope	Beijing Zhongtian Guangzheng Technology Co., Ltd.	TS-39NK	Used for performing mouse cardiac transplantation surgery
Cyclosporine A	Novartis Pharmaceuticals (Switzerland)	H20100673	Used for inhibiting TCMR in recipient mice
FITC anti-mouse IgG Antibody	Bio Legend (USA)	406001	Used for flow cytometric quantification of DSA-IgG levels
Flow cytometer	Becton Dickinson, USA	BD FACScalibur	Used for assessment of serum DSA levels in recipient mice
PE anti-mouse IgM Antibody	Bio Legend (USA)	406507	Used for flow cytometric quantification of DSA-IgM levels
Small animal gas anesthesia system	Anhui Zhenghua Bio-Instrument Equipment Co., Ltd.	ZH-MZJ	Equipped with isoflurane for general anesthesia
Vascular Bulldog Clamps	ROBOZ SURGICAL INSTRUMENT CO.	RS-5481T	Used to block blood flow during cardiac transplantation