13. Recombinant Octameric Hemoglobins as Resuscitation Fluids in a Murine Model of Traumatic Brain Injury Plus Hemorrhagic Shock

Three recombinant octameric mutants of human normal adult hemoglobin (Hb A), rHb (αN78C), rHb (αN78C/L29F), and rHb (αN78C/L29W), were expressed in our Eschericia coli expression system and purified. They were used as resuscitation fluids in our unique mouse model of traumatic brain injury (TBI) combined with severe hemorrhagic shock (HS). A sulfhydryl group was introduced onto the surface of the α-subunits of rHb A by substituting Asn78 with cysteine. The rHb (αN78C) form octamers by linking the two tetramers with 2 intermolecular disulfide bonds. Nuclear magnetic resonance (NMR) spectroscopic studies indicate that rHb (αN78C) has the same quaternary and tertiary structures as those of Hb A. Furthermore, the oxygen-binding activity (as measured by P ₅₀ ) and the cooperativity of the oxygenation process (as measured by the Hill coefficient) of this mutant have not been altered compared to Hb A. The Leu29 residue on the α-subunits of the octamers was then mutated into either phenylalanine (F) or tryptophan (W) to yield rHb (αN78C/L29F) and rHb (αN78C/L29W), respectively. Compared to Hb A, rHb (αN78C/L29F) has high- while rHb (αN78C/L29W) has low-oxygen affinity. Both mutants are cooperative in their oxygen-binding properties, but lower than that observed for Hb A. They maintain their quaternary structure as those of Hb A, but exhibit perturbation of their tertiary structure at or near the heme pockets as detected by NMR measurements. Although all octameric rHbs would be expected to have reduced nitric oxide (NO) binding based on their size, rHb (αN78C/L29F) and rHb (αN78C/L29W) may have a further reduction in NO binding as a result of introducing a bigger aromatic amino acid residue at the distal heme pocket of the α-chain. These three rHbs were used as resuscitation solutions in mice after TBI combined with HS. TBI was induced by a controlled cortical impact (CCI) to the left parietal cortex. Blood was then withdrawn (2.4 mL/100 g body weight) over 15 min to induce HS. A pressure-controlled model was used with the mean arterial pressure (MAP) maintained at 25–27 mm Hg for an additional 20 min. At the end of the HS Phase (35 min total), i.e., the beginning of Pre-Hosptial Phase, lactated Ringers (LR) or rHb (120 mg/mL) solutions were administered to the mice at 2 mL/100 g. Additional LR or rHb solution was given if needed at 1 mL/100 g to maintain the MAP at > 70 mm Hg for the next 90 min. The shed blood was then returned simulating definitive care with transfusion in a “Hospital Phase”, lasting 15 min. The mice were then recovered, returned to cages and observed for 24 h before sacrificing for neuropathology. A marked difference in the fluid requirements was observed between the LR and the rHb groups. At the end of the Pre-Hospital Phase, the LR group received >4 times more resuscitation fluid (21.5 ± 0.75 mL/100 g) than the rHb groups (5.0 ± 0 mL/100 g; P < 0.05), while the arterial Hb level in rHb groups were ~3 g/dL higher than that of the LR group. More importantly, the LR group exhibited persistent refractory hypotension during the Pre-Hospital Phase. The MAP of rHb groups stayed near baseline level over the entire Pre-Hospital Phase and Hospital Phase. However, an initial elevation in MAP above baseline followed by gradual diminution was observed only for the rHb (αN78C) group (P < 0.05). Brain tissue oxygen (PbtO₂) levels in the hippocampus ipsilateral to the site of CCI did not differ significantly between groups, although the LR group showed deterioration in PbtO₂ during late Pre-Hospital Phase. Numerically, the normal oxygen affinity rHb (αN78C) group and the high oxygen affinity rHb (αN78C/L29F) groups exhibited the highest and lowest PbtO₂, respectively. Surprisingly, the high oxygen affinity rHb conferred a neuroprotective effect in the CA1 region of the selectively vulnerable region of the hippocampus vs. all other groups. No difference in neuronal survival was seen between groups in the CA3 hippocampus. Our present results suggest that these novel octameric rHbs have the potential to develop into a small-volume resuscitation fluid for treatment of TBI combined with HS.