Analysis of Esterified F- and D/E-Ring Isoprostanes and Neuroprostanes, and Lipid-Soluble Antioxidants in Brain Stem Tissues from Rats Following Experimental Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) resulting from aneurysmal rupture is a common type of hemorrhagic stroke in humans. Vasospasm following SAH may lead to delayed ischemic neurological deficit (DIND), which is the major cause of poor outcome after surgery in patients. Hemoglobin in the subarachnoid space after hemorrhage is involved with the occurrence of vasospasm and oxidative stress, but there was no convincing evidence of oxidative damage until our recent publication (Free Radical Biology & Medicine 40: 1466, 2006). We have found that levels of CSF F2-isoprostanes (F2-IsoPs) were positively correlated with poor outcome of patients. F2-IsoPs has become the golden marker of lipid peroxidation in vivo. It is derived from arachidonic acid following lipid peroxidation reactions. It is initially generated as esterified from on phospholipids and then released into surrounding body fluids by certain enzymes. Moreover, the proportion of D/E-ring over F-ring IsoPs will be increased when reducing substances are decreased. Certain isomers of F2-IsoPs and E2-IsoPs have potent vasoconstrictive activity as PGF2. Furthermore, F-ring and D/E-ring neuroprostanes (NPs) are generated from peroxidation of docosahexaenoic acid with similar mechanisms. NPs are indicators of oxidative damage from neuronal cells as DHA is enriched in neurons. However, currently only the lab of the principle investigator of this project has the complete technique to analyze free F2-IsoPs and F4-NPs in biological fluids by using gas chromatography/mass spectrometry with negative-ion-chemical-ionization function (GC/NICI-MS) in Taiwan. In this newly 3-year proposal, we will first need to establish the platforms to analyze D/E-ring IsoPs and NPs; and esterified IsoPs and NPs. Secondly, we will investigate whether levels of oxidative damage would be increased before and after vasospasm following experimental SAH in rats with both single-injection and double-injection models. We will measure esterified forms of F2-IsoPs, D2/E2-IsoPs, F4-NP, and D4/E4-NPs in brain stem tissues at different time points after initial injection of autologous arterial blood. On the other hand, we will detect levels of different tocopherols and coenzyme Q by CoulArray HPLC in the same brain stem tissues in order to obtain complementary information on the changes of lipid-soluble antioxidants that can suppress lipid peroxidation. Finally, we would like to separately study whether vitamin E (α-tocopherol), an inhibitor of lipid peroxidaiton, and EUK-189, a superoxide dismutase/catalase mimetic, can suppress oxidative damage, vasospasm, and neuronal damage induced by experimental SAH in rats at the same time. Taken together, this study may provide more solid evidences on the significance of different markers of lipid peroxidaiton or oxidative damage in SAH-induced vasospasm using a rat model. This study may also establish an important animal model to screen effective antioxidants that can suppress experimental SAH-induced vasospasm and oxidative damage, which may be helpful in developing strategies to improve therapy of SAH patients in the future.

Project ID:PC9902-0466
External Project ID:NSC97-2320-B182-012-MY3