Hypotension from hemorrhage complicating traumatic brain injury (TBI) contributes to secondary injury and poor outcome, including both morbidity and mortality. The deleterious effects of hypotension have been demonstrated in both civilian (1
) and military settings such as blast injury (2
). Effective resuscitation of combined TBI and hemorrhage is essential to improving outcomes in the increasing numbers of individuals exposed to these combined injuries, particularly, in military theaters abroad (3
Traditional pre-hospital strategies for TBI resuscitation involve the administration of isotonic fluids, such as Lactated Ringer’s (LR) solution, or colloids, such as Hextend (HEX) (the civilian and military standards of care, respectively) after injury to restore blood pressure, and potentially, cerebral perfusion pressure. However, large volumes of resuscitation fluid may exacerbate cerebral edema (4
), and fail to increase oxygen delivery. In addition, these solutions fail to specifically target deleterious cascades associated with ischemia reperfusion injury. In the pre-hospital setting, hemoglobin (Hb) based oxygen carriers (HBOCs) may offer specific advantages over crystalloids or colloids as a resuscitation fluid in the setting of hemorrhagic hypotension after TBI. HBOC resuscitation may allow for volume expansion using small amounts of fluid with the potential advantage of restoring oxygen delivery to the injured brain—which exhibits markedly increased metabolic demands and excitotoxicity early after TBI (5
). However, human clinical trials have shown increased morbidity and mortality with the use of HBOCs (6
). Current generation HBOCs and cell-free Hb scavenge nitric oxide (NO), resulting in vasoconstriction and decreased oxygen delivery to myocardium and other vascular beds (7
). This could also impair posttraumatic cerebral blood flow (CBF), since NO levels and endothelial nitric oxide synthase (eNOS; NOS1) activity are compromised early after TBI in injured brain regions (8
). Additional putative deleterious side effects of HBOCs have also been suggested. Winslow (9
) proposed that increased oxygen delivery caused by free Hb juxtaposed to vascular endothelium causes local hyperoxia with coupled vasoconstriction which could further impair CBF in injured brain. Similarly, HBOCs and cell-free Hb can also directly contribute to oxidative stress in both the microcirculation and parenchyma. Specifically, extravasation of cell free Hb into tissue could result in direct cytotoxiciy and this could be of considerable importance in TBI, since vascular disruption occurs and classic studies have shown that cell free Hb is highly neurotoxic in cell culture (10
Chemical modification of the Hb structure has been studied to minimize the harmful side effects of HBOCs. Nitroxyl- groups have well-known antioxidant effects including potent superoxide dismutase (SOD) mimetic activity, along with other favorable effects on oxidative stress, such as limiting autoxidation (12
). Polynitroxylation of Hb also creates redox coupling between the convalently labeled nitroxides and the heme iron to provide additional catalase mimetic activity (13
). Pegylation (i.e., adding polyethylene glycol [Peg] moieties to the Hb molecule) provides several potential favorable effects including limiting the Hb from direct interaction with the endothelium—attenuating oxygen mediated vasoconstriction (14
) and enhancing nitrite reductase activity—with increased NO production and blunted vasoactivity (15
). Pegylation also adds a “super-colloid” effect to an HBOC, due to the potent water scavenging effect of Peg moieties (Personal communication, A. Abuchowski, PhD), and prolongs HBOC half life. Polynitroxylated-pegylated Hb (PNPH) is a novel bovine HBOC developed by SynZyme Technologies that uses Prolong Pharmaceutical’s Peg-Hb as the starting material, and thus has both of these chemical modifications. Specifically, it has ~14 nitroxyl radicals (nitroxides) and 8–10 Peg moieties (5KDa molecular weight each) per Hb tetramer molecule. It is prepared as a 4% Hb solution.
We recently published (16
) details of a mouse model in which a mild controlled cortical impact (CCI) injury to brain is followed by 90 min of volume-controlled hemorrhagic hypotension resulting in an exacerbation of neuronal death in the vulnerable CA1 hippocampus vs that seen in either CCI or hemorrhage alone. Blood flow is compromised in the hippocampus beneath the contusion during the hemorrhage. The model is perfectly suited for exploratory evaluation of novel HBOCs in the setting of TBI resuscitation in that it is allows for the assessment of acute hemodynamics, and long term outcome including neuropathology. Mortality rates in the model with standard resuscitation solutions range between 25 and 30%. Unlike large animal models, novel therapies can be explored both with extremely small volumes of novel resuscitation fluids and with relatively limited expense.
We tested two hypotheses: 1) PNPH is a unique non-neurotoxic HBOC in neuronal culture and is neuroprotective in in vitro neuronal injury models, and 2) Resuscitation with PNPH would require less volume to restore mean arterial blood pressure (MAP) than LR or HEX and confer neuroprotection in a mouse model of TBI plus hemorrhagic hypotension. In the present communication, we present results that show that PNPH is a unique neuroprotective Hb both in vitro and in vivo and that support its further development and evaluation as a neuroprotective HBOC for small volume pre-hospital TBI resuscitation.