Optimization of a hibernation-based small-volume resuscitation fluid

Abstract

<bold>Background:</bold> Hemorrhagic shock is the number one cause of preventable deaths after trauma. Hibernation exemplifies a physiological state in which blood flow is reduced to a magnitude comparable to that of hemorrhagic shock. However, hibernators are naturally shielded from the insults that a similar reduction in blood flow would cause in a non-hibernating mammal. Our laboratory previously published a small volume resuscitation fluid based on hibernation physiology:BHB/M. It has three main components: 1) 4 M BHB, 2) 43 mM melatonin, and 3) 20% DMSO. Only the indicated concentration of each component of BHB/M has been previously tested. For that reason, worked towards the optimization of the composition and delivery of BHB/M in order to enhance survival in a rat model of hemorrhagic shock.<bold>Methods and Results:</bold> Previously, BHB/M was given as a 1 ml/kg bolus followed by a 100 µl/hr slow infusion. BHB/M was administered as either a single bolus or a bolus plus slow infusion in acutely operated rats. There were no statistical differences (p>0.05) in mean survival times when comparing the bolus only (mean survival 496.67 ± 314.59 min. n=6) to the bolus plus slow infusion (mean survival 149.20 ±142.71 min. n=5) protocol.Two separate dose-ranging studies were conducted for both BHB and Melatonin. In the BHB dose-ranging study, BHB was administered at either a 4 M,2 M, or 0.4 M concentration in conjunction with 4.3 mM melatonin and 10% DMSO. 10-day mean survival showed a dose-dependent trend where the higher the concentration of BHB infused the longer the rats survived (4 M BHB, 7.38 ± 1.75 days; 2 M BHB, 5.25 ± 2.22 days; 0.4 M BHB, 2.07 ± 2.05 days). In the melatonin dose-ranging study, melatonin was administered at either a 4.3 mM, 0.43 mM 0.0043 mM, 0.000043 mM, or 0 mM concentration in conjunction with 4 M BHB and 2% DMSO. An osmolarity control composed of 4 M NaCl and 0.000043 mM melatonin in 2 % DMSO was also included. Administering 4 M BHB without melatonin resulted in very low mean survival times (4.38 ± 1.42 days); the same was true when infusing 0.000043 mM melatonin with 4 M NaCl (4.58 ± 1.42). All treatments containing both 4 M BHB and melatonin, regardless of concentration, resulted in mean survival times of ~7.5 days.A large-volume experiment was conducted in order to compare isotonic BHB/M to the standard of care (LR). Rats subjected to 60% blood loss were infused with either LR, LR plus 4.3 mM melatonin, 140 mM BHB with 1.5x10-6 mM Mel, or 140 mM BHB with 4.3 mM Mel. LR and LR plus 4.3 mM melatonin had statistically higher (p<0.05) mean survival times (7.35 ± 1.59 days and 6.73 ± 1.57 days, respectively) than 140 mM BHB with 1.5x10-6 mM Mel (2.08 ± 1.18 days).<bold>Conclusions:</bold> BHB/M can be administered as a single 1 ml/kg bolus; following the bolus with a 100 µl/hr slow infusion is not necessary to obtain a maximum survival benefit. BHB must be administered at a 4 M concentration as there is a dose-dependent trend in which the lower the concentration of BHB administered the lower the percent survival to 10 days. Melatonin provides therapeutic effects at very low concentrations evident by the survival observed when administering a solution containing melatonin at a concentration a million-fold lower than previously published. Furthermore, melatonin is essential for survival since 4 M BHB without melatonin had a considerably reduced survival rate. A large volume dilute BHB/M is not a viable alternative to the standard of care.