Introduction: Dysfunction of the cerebrovasculature, manifested by poor reperfusion and loss of autoregulation, is a feature of ischemia and traumatic brain injury (TBI). Oxidative stress is implicated in this effect. Conventional antioxidants have not proven clinically effective. Nanomaterials are an emerging class of antioxidants with potential advantages including quenching of oxidative radicals without need for enzymatic transformation.
Objectives: We tested whether the carbon nanomaterials, poly(ethylene gylcol)-functionalized hydrophilic carbon clusters (PEG-HCCs) are antioxidants and determined their effect on the cerebrovasculature following mild experimental TBI and hypotension.
Methods: HCCs were generated by treating single wall carbon nanotubes with oleum and nitric acid. HCCs were functionalized with PEG via coupling to carboxcylic acids. The ability to quench superoxide anion (SO) was determined in solution and b.End3 cultured brain endothelial cells after administering the electron chain transport inhibitor Antimycin A (AntA) using dihydroethidium (DHE) fluorescence flow cytometry. In-vivo studies were performed in Long Evans rats following mild TBI (3m/s; 2.5 mm deformation; 80 ms duration) and 50 mins. hemorrhagic hypotension followed by resuscitation with Lactated Ringers ( Figure : Resus ) then by shed blood ( Hospital ). Treatment with 2 mg/kg PEG-HCCs in 1 mL or PBS was initiated prior to the Hospital phase. Laser Doppler perfusion (LDF) was measured to 6 hours post-TBI.
Results: DHE fluorescence induced by AntA was eliminated by post-treatment with PEG-HCCs (2-4 mg/L; 15 min post-AntA). At that concentration, there was no innate toxicity in cells determined by clonogenic and trypan blue assay. Only pre-treatment with a 10X excess of SO dismutase (SOD) achieved comparable DHE effect. PEG-HCC post-treatment improved cell survival after a higher dose of AntA, to 65% of baseline compared to 23% for SOD. In-vivo following TBI, hypotension reduced relative LDF to approximately 30% ( Fig ). Administered just prior to shed blood nanotubes restored LDF to 100% of baseline while the vehicle group remained significantly lower at 56%. LDF declined in all groups over time.
Conclusions: PEG-HCCs are biocompatible with b.End3 cells and rapidly protected these cells from oxidative stress. PEG-HCCs at a clinically realistic time point improved cerebrovasculature dysfunction post-TBI/hypotension. The duration of effect is consistent with blood half life after a single dose. Longer term dosing studies are underway to establish the effect on outcome and reperfusion injury.