Establishment and Characterisation of a Murine Model of Brain Death and Syngeneic Renal Transplantation.

Abstract

Aim: Donor brain death (BD) adversely affects organ quality. It has been associated with more rapid rejection of renal transplants and may also compromise the ability to achieve tolerance. Here, we aimed to establish a model of donor brain death and subsequent renal transplantation in mice. Methods: C57BL/6 mice were anaesthetised using Ketamine/Xylazine. A burr-hole was created, through which a 2F Fogarty balloon catheter was introduced. Pressure-controlled mechanical ventilation was commenced via a tracheostomy. Ventilator settings included 5cmH2O PEEP. Mean arterial pressure (MAP) was measured via a femoral cannula. MAP, HR, core temperature and O2 saturation were continuously monitored. BD was induced by controlled inflation of the balloon with 80-100μL at 5μl/min, and was confirmed by the absence of spontaneous respiration after ventilator disconnection. MAP was maintained ≥40mmHg for >3 hours using boluses of 0.25mL gelofusine iv. Samples were collected at 30 mins, 1 and 3 hrs after the induction of BD. Kidneys from some mice at 1 hr post-BD were grafted into syngeneic recipients. Samples were collected at d1 and 4 post-Tx. Serum HMGB1, S100B and cytokines were quantitated, and renal histology, cellular infiltration and gene expression determined. Results: A typical Cushing response was observed with a spike in MAP from 60mmHg to over 90mmHg, accompanied by tachycardia. Tachycardia persisted, whilst MAP slowly declined. Serum S100B (10163±2387pg/ml cf 1731±557, p=0.03), HMGB1 (25.0±3.2ng/ml cf 8.1±1.2, p<0.01), cytokines, and cytokine gene expression in kidneys were substantially increased by 1 hour after BD induction, notably IL-6 (105 pg/ml cf 18±15, p<0.01), KC (8830±1170pg/ml cf 97±29, p=0.02), and IL-1β (150±21pg/ml cf 3±0, p=0.02). The extent of neutrophil and macrophage infiltration was dramatically increased in kidneys from BD donors on day 4 post-Tx, compared with that in ctrl syngeneic grafts (nΦ 25.7±6.0/HPF cf 1.4±0.3, p<0.01 and F4/80 65.1±9.1/HPF cf 23.7±6.2, p<0.01). Gene expression for RAGE, TLR4, CXCL10, MCP-1, IL-6, TNF-α, TGF-β, IL-1β and HSP2 was considerably greater in kidneys from BD donors. Conclusions: A mouse model of brain death and subsequent renal transplantation was successfully established. Physiological and biochemical changes parallel those observed in clinical transplantation.