Azulenyl nitrones: colorimetric detection of oxyradical end products and neuroprotection in the gerbil transient forebrain ischemia/reperfusion model

We present analytical and neuroprotective data on a unique spin trapping agent derived from a novel chemical class known as an azulenyl nitrone (AZN). Based on Colorimetric properties, AZN was used to assess the formation of free radicals in a bilateral carotid occlusion (BCO) model in gerbils by monitoring the conversion of the nitrone to the aldehyde in affected tissue. In addition, AZN was tested as a neuroprotectant in this model regarding the preservation of CA1 pyramidal cells of the hippocampus following transient ischemia/reperfusion. AZN was electrochemically oxidized to give the aldehyde using an HPLC system with on line electrochemical oxidation. The oxidation potential associated with a 50% loss of AZN occurred at about 600 mV (half-wave potential versus palladium electrode). The major product detected as AZN oxidation occurred in an aqueous methanolic medium was the corresponding azulenyl aldehyde. Oxidation of AZN was inversely related to the formation of the aldehyde. Based on this test, we considered the in vivo conversion of AZN to aldehyde to be a measurement of oxidative stress in tissue. Results show that 0.3% of hippocampal AZN was converted to aldehyde in animals treated as shams. However, in gerbils subjected to a 7-min ischemic insult plus 7-min reperfusion, the conversion rate was about 3 times higher at 1.0%. In this model, surviving CA1 hippocampal neurons were counted from gerbils that were subjected to 7 mins of BCO followed by 5 days of reperfusion. In sham animals, about 89 cells were counted in a selected field of CA1 neurons. With injury, only 27 cells on average survived (70% loss) and were counted from this selected field. Under similar conditions and AZN treatment, 57 cells survived (36% loss). We conclude, therefore, that the demonstrated neuroprotection occurs because AZN neutralizes radicals which contribute to neuronal damage following ischemia/reperfusion.