Although the components affected by cerebral ischemia include glutamate excitotoxicity, increased nitric oxide levels, calcium ion movement, and eicosanoid synthesis, a common element in the mechanisms of toxicity results from disruption of oxidant homeostasis. The hypotheses to be tested in this project are that a component of the pathology of the lesions present in ischemia is due to disruption of GSH and antioxidant homeostasis and that neurotrophic- based manipulation of GSH and antioxidant homeostasis may ameliorate these pathological consequences. The neurotrophins to be studied here are nerve growth factor (NGF) and brain-derived neurotrophic factor BDNF. They differentially enhance cellular defenses by increasing antioxidant activity and pyridine nucleotide metabolism, critical to neuronal resistance to and recuperation from injury in vitro and in vivo. Ischemia will be studied in a rodent model relying on middle cerebral artery occlusion (MCA) and all measurements will focus on the hippocampus and basal forebrain as sites of plasticity in cNS associated with glutamate action, nitric oxide synthesis, calcium fluxes and stress responses. The hippocampus is the site in the central nervous system [CNS) with the highest levels of neurotrpphin synthesis and the basal forebrain is a site of synthesis of p75 NGFR and trk receptors. We will monitor by genetic resonance imaging [MRl) the effects of blood flow restriction on infarct size and by 31P- NMR spectroscopy the fate of active phosphate species and pH. We will determine effects of MCA occlusion on glutathione peroxidase (GSH-Px), glutathione reductase (GSH- Rx), and y-glutamyl cysteine synthetase [GCS), important enzymes for GSH redox and synthesis, shown to be regulated by NGF in vitro. We will ensure levels of these enzyme activities spectrophotometrically and the mRNAs by Northern analysis, RNA protection assays, and reverse transcriptase polymerase chain reaction, with suitable controls. We will monitor levels of key substrates and cofactor, such as GSl-l, GSSG, and NADPH. We will determine the effects on neurotrophic and neurotrophic receptor expression in ischemia and monitor these by immunohistochemistry and in situ hybridization. We will introduce NGF and/or BDNF stereotaxicly or transplant cell lines retrovirally transfected with the NGF or BDNF gene before and after the stroke lesion. While the animal model used is likely to involve multiple physiological responses in CNS, our working hypothesis is that the aspects of oxidant homeostasis here studied constitute a common element vital to cell survival and are to some extent regulated by neurotrophins. These studies will provide insights into the mechanism of stroke pathophysiology and suggest new therapeutic approaches.
Showing the most recent 10 out of 15 publications
