Aorta Transplantation Model On Mice For The Study Of Naturally Occurring Antibody In The Context Of Chronic Allograft Vasculopathy

Backgrounds:Short-term attrition rates of implanted grafts have significantly declined over the past decades due primarily to the use of potent T cell immunosuppressive therapies.Unfortunately,long-term allograft survival rate has remained unresponsive to current treatments since the machanisim of chronic rejction has not been deciphered completely.Allograft vasculopathy seems to be a common reason for graft loss in all vascularized solid organs.Antibody mediated rejection,donor specific antibody(DSA)to be specific,is widely acknowledged to be associated with allograft vasculopathy and,in fact,the leading cause of late graft loss in the context of heart and kidney transplantations.While our preliminary clinical study demonstrated that pre-transplant naturally occurring antibodies(Nabs)level(generated by innate B cell,B1 B cells in mice),independent of DSA,is associated with lower long-term kidney allograft survival rate.We sought to demonstrate the correalation between serum Nabs and chronic allograft vasculopathy by means of aorta transplantation on mice.Methods and materials:1.Inducing endogenous Nabs: B/6 recipient mice will be immunized with UV irradiated syngeneic apoptotic thymocytes once/week over a period of 4 weeks.The development of Nabs will be monitored every week by assessing the serum reactivity to apoptotic Jurkat cells by flow cytometry;2.Developing a mouse aorta transplant model: In order to facilitate the surgical technique by using the “cuff” technique,the model consists in harvesting a segment of the thoracic aorta of donor mice,applying the donor aorta into the “cuff”,and replacing the infrarenal abdominal aorta with the fashioned donor aorta in a heterotopic manner.The severity of allograft vasculopathy will be assessed appropriately.3.Grouping up: Set 4 experimental groups as follows: 1)Allogeneic/Immunized;2)Allogeneic/Non-immunized;3)Syngeneic/Immunized;4)Syngeneic/Non-immunized.Harvest transplanted graft aorta for histology studies 6 weeks after successfully performing operations as designed.Results:1.FSCS analysis of mice serum testified the gradual development of Nab concentration and its consistency will stay until the endpoint of the study(4-6 weeks).Preexistence high Nab mouse model is therefore established.2.The “cuff” technique is proven to be a better method compared to conventional suture-based anastomosis technique.Numeric variables including intimal proliferation index(I/M),occlusion percentage(%)and cell number in media/mm2 are found to be adequate enough to evaluate the severity of allograft vasculopathy.3.Allogeneic donor aorta developed neointima as much as I/M=0.96±0.13,Occlusion %=24.8±3.9%,while allogeneic aorta exposed to high level of endogenous Nab showed an undeveloped lumen abnormality I/M=0.13±0.04,Occlusion %=4.4±2.0%.T tests between these groups for I/M and occlusion % are significant(PI/M =0.004,Pocclusion =0.0108).Sign of vasculopathy with the effect of preexisting Nabs was limited progressively that it was statistically comparable to that of Syngeneic/Non-immunized allograft aorta(Allogeneic/Immunized: I/M=0.13±0.04,Occlusion %=4.4±2.0%;Syngeneic/Non-immunized: I/M=0.07±0.04,Occlusion %=5.8±0.2%;PI/M=0.3170,Pocclusion=0.6352).Conclusion:Under the exposure to high level of endogenous Nabs,the progression of intimal proliferation will be vastly limited in the context of mouse allogeneic aorta transplantation.