Objectives: The goal of this 5-year grant is to assess efficacy of this nanotechnology-based dual inhibitory strategy to extend cold-ischemia time and regulate inflammatory response after limb transplantation in rats. Specifically, we will evaluate the influence of treating the donor, the excised limb graft within preservation solution, or the recipient with the LSNA nanoparticle in our in vivo rat model of limb transplantation. The primary endpoint is time to recovery of sensory and motor function in recipient rats of composite grafts treated with the inhibitory nanoparticle versus control. Secondary endpoints are: 1) degree of immune dampening by lymphocyte profiling conducted by our immune monitoring core post reperfusion, 2) analysis in reduction of pro-inflammatory cytokines after reperfusion by Luminex assay and 3) histologic tissue assessment at sacrifice of recipient animals. The first year will complete the standardization of the limb transplant in rodents. Years 2 will refine strategies to deliver the LSNA to the graft. Year 3 will assess the functionality of non-targeting control LSNAs within the limb transplant model. Years 4-5 will characterize the dual targeting inhibitory LSNAs in the model using various treatment strategies (e.g. pretreatment of donor, graft treatment during cold storage, and treatment of the recipient animal after transplantation. Additional studies during this time will assess the biodistribution of LSNAs within recipient rats after treatment. Methods: Hind limbs are recovered by the rodent microsurgery core facility from donor rats by ligating femoral vessels just below the inguinal ligament. The femur is divided at mid-thigh. The prepared limb is perfused through the femoral artery with cold University of Wisconsin (UW, 4°C) solution containing the dual inhibitory or control nanoparticle and immersed in cold perfusate for 2h or 4h. The donor limb is implanted into syngeneic recipient rats using a needle as an intra-medullary pin. Muscles are approximated. The venous anastomosis and arterial anastomosis are completed under microscopic magnification with 11-0 nylon sutures. The sciatic nerve is repaired. No immobilization or systemic anticoagulation is required. Expected Results: Based upon our preliminary studies using isolated cells, we anticipate that duel TLR inhibitory nanoparticles will inhibit the innate immune response following IRI after limb transplantation in rats. We are uniquely suited to carry out these studies because of our highly trained microsurgical core and the ability of our immune monitoring team to track immune response following transplantation. The use of a pre-clinical animal model to develop strategies to prevent rejection in limb transplantation is extremely valuable to the field due to the limited number of human patients. The ability to influence the immune response via novel technologies is the next step in tissue transplantation to improve outcomes, minimize rejection and prevent secondary injury to other organs that are impaired as a result of chronic exposure to immunosuppressants. The experimental results in this study using limb transplantation will have broader implications beyond extremity transplant alone where the technology developed through this study will also be applicable to clinical transplantation for end-stage diseases of vital organs (heart, kidney, liver) commonly seen in the veteran community.
|Effective start/end date||4/1/16 → 3/31/22|
- Northwestern Memorial Hospital (Agreement 6/5/20)
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