The Role And Mechanism Of DMSO In Cryopreservation And Alleviating The Reaction Of Ischemia Reperfusion

Backgroud:As the only radical treatment for terminal-stage organ failure,organ transplantation had made significant progress in the past and will become one of the main directions of medical development.Organ preservation has a pivotal position in the process of transplantation.The research on the organ preservation solution has achieved new breakthroughs constantly since the idea of organ preservation in vitro was proposed by scientists in 1930 s.Followed by HTK solution,Celsior solution,Polysol solution,SCOT solution,CZ-1 solution reported by Chinese scholars and other solutions,the University of Wisconsin(UW)solution was developed successfully and applied to the clinical medicine until 1988.Because UW solution can be used to preserve various organs effectively,it becomes the“Gold Standard” for organ preservation solutions.Although the clinical has begun to explore and apply a new series of organ preservation techniques,the position of cryopreservation is still dominant in organ preservation,and organ preservation solutions are the core of cryopreservation.The development of the organ transplantation field has been restricted by the shortage of donors currently.In order to solve this problem,a large number of marginal donors were used clinically,which resulted in an increasingly higher requirement for organ preservation solutions.As a sulfur-containing organic compound,Dimethyl sulfoxide,(DMSO)is a kind of colorless and odorless transparent liquid at room temperature,miscible with water and soluble in ethanol,propanol,benzene,chloroform and many other organic matters.In addition to being used as hydroxyl radical scavenger and cryoprotectant,DMSO is also verified suitable for the treatment of many diseases,such as curing prostatitis,gastrointestinal diseases,inflammatory diseases and improving the renal functions of patients with kidney amyloidosis caused by Crohn’s disease.Moreover,the immune modulating function of DMSO in humans was reported by Kloverpris et al in 2010,and itwas reported by Gu-Jiun Lin et al that DMSO could act on regulatory T cells.In light of the above-mentioned anti-inflammatory and antioxidant functions of DMSO,we conducted a research on whether DMSO could be used to extend the preservation time of transplantation donors through mice cervical heterotopic heart transplantation model,from which a conclusion was drawn that DMSO could significantly extend the cold preservation time of C57BL/6 mice heart.In order to clarify this protective effect,a study was carried out on the functions of DMSO for the myocardial change and ischemia reperfusion injury in the process of cardiac cold preservation,so as to provide experimental data for the illustration of the role and mechanism of DMSO in cryopreservation and alleviating the reaction of ischemia reperfusion.Part One: The Establishment of Mice Cervical Heart Syngeneic Allograft Model and the In-vitro Study on the Effects of DMSO on the Mice Heart Cryopreservation Time and DamageObjective: To establish a cervical syngeneic heart allograft model of mice and to study the effects of DMSO on the mice heart cryopreservation time and damage through an in-vitro research.Method: 1)Cervical heterotopic heart grafting was conducted on mice,among which six-week-old C57BL/6 mice with matched weight and gender were selected as the donors,while 8-week-old C57BL/6 mice with matched weight and gender were selected as the recipients.The hearts were stored in normal saline with different concentrations of DMSO,to study the cold-ischemia preservation time limit of mice hearts(saline was used in the control group).The judgment standard was the grafts’ survival.2)The cold-ischemia preservation time of mice hearts and myocardial injury results were researched under optimal DMSO concentration through detecting the myocardial enzyme spectrum and inflammatory factors,and histological detection.Result: Compared to the control group which was preserved in saline,the DMSO-treated hearts of donors showed different degrees of increase in cryopreservation time and 0.5% DMSO concentration was optimum for preservation;HE staining showedthat the myocardial edema,necrosis and other symptoms of the mice in the experimental group were significantly alleviated compared with the control group;according to the immunohistochemical results,the myocardial apoptosis in both the experimental group and the control group showed no significant difference before reperfusion,but differences began to appear after reperfusion with increase in DMSO concentration.Conclusion: DMSO can extend the cold preservation time of grafts and alleviate the ischemia reperfusion injury.Part Two: Researching the Function and Mechanism of DMSO for Grafts’ Reperfusion Injury by Using Syngeneic Transplantation ModelsObjective: To study the function and mechanism of DMSO on grafts’ reperfusion injury.Method: 1)The best concentration of DMSO was selected to preserve the hearts for six hours(normal saline was used for the control group),after the syngeneic transplantation was conducted: serum myocardial enzyme spectrum,MDA and inflammatory factors were tested by means of ELISA in 4 hours and 12 hours after the transplantation.2)The expression of grafts’ cytokine was detected through RT-PCR.3)The expression of NF-κB、JNK-1、ERK1/2、p38MAPK、BAX、Bcl-2、Caspase-3 and Caspase-9after reperfusion was tested by Western-blot detection.4)The primary cardiac myocytes of C57BL/6 mice were cultured in vitro,and anoxia and reoxygenation experiments were carried out in the experimental group after applying the optimum concentration of DMSO,and then the expression of NF-κB、JNK-1、ERK1/2、p38MAPK、BAX、Bcl-2、caspase-3and caspase-9 of myocardial cells was tested by Western-blot detection.Result: After the DMSO-treated mice hearts went through reperfusion,the TNF-αand IL-1β became significantly lower than the control group,but the IL-6 showed no significant difference from the control group.At the same time of reperfusion,the levels of TNF-α,IL-1β and IL-6 rose along with the extension of preservation time.Compared to the control group,the various indexes of myocardial enzyme spectrum of the recipient mice showed different degrees of decline;the result of RT-PCR was the same as with theone of ELISA;the expression of the phosphorylated Bcl-2 in the experimental group was higher than the one in the control group,while the expression of p-p65、p-JNK-1、Bax、cleaved caspases-3 and cleaved caspases-9 was lower than the one in the control group;the results of the in-vitro and in-vivo experiments were the same.Conclusion: DMSO can alleviate grafts’ ischemia reperfusion injury after cold preservation via the NF-κB pathway and reduce the apoptosis mediated by mitochondria.