Modulation of cerebral blood flow is not the mechanism of neuroprotection in transgenic mice over expressing human extracellular superoxide dismutase (EC-SOD)
Introduction: Transgenic mice over expressing human EC-SOD (5-fold increase in brain) that have received a 90 min filament occlusion of the middle cerebral artery (MCAO) have a 27% decrease in infarct volume compared to wild type controls (1). Superoxide (SO) is one of the primary scavengers of nitric oxide (NO), a reaction that is only diffusion limited. Therefore, scavenging of SO by EC-SOD may result in increased levels of NO (2). NO stimulates vascular smooth muscle relaxation. In brain this results in cerebrovascular dilatation and increased cerebral blood flow (CBF). This increase in CBF may be the mechanism of neuroprotection in transgenic EC-SOD mice. Methods: Transgenic mice were generated by injecting nuclei of fertilized oocytes from B6C3F1 female mice with the cDNA of human EC-SOD containing a β-actin promoter. Mice carrying the transgene were identified by a probe and Southern blot analysis. Over expression of EC-SOD in the brain of transgenic mice was verified by SOD activity analysis. Fasted male EC-SOD (n=7) and wild type (WT; n=10) mice were anesthetized with halothane and allowed to breath spontaneously. Animals were surgically prepared for MCAO. Rectal temp was servo-controlled at 37.0°C. After a 20 min interval of MCAO, 5 μCi 14C-labeled iodoantipyrine was infused i.v. over 65 sec via ramp infusion. Simultaneously, twelve 10 μl discontinuous arterial blood samples were collected for later determination of arterial 14C activity. Upon completion of isotope infusion the animals were decapitated and brains frozen in 2-methylbutane (-40 °C). Brains were sectioned (20μ), autoradiographs made and then digitally imaged and analyzed. Arterial blood sample radioactivity was determined by liquid scintillation counting using an external quench correction. Values are mean±sd and statistical analysis by unpaired Student's t test. Results: Volume of brain at risk for infarct was defined as CBF less than 25ml/100g/min. Volume at risk was similar between the two groups (mm3; cortex: WT=51±15, EC-SOD=47±9, p=0.65; subcortex: 39±16, 37±17, p=0.81). Total hemispheric volumes were similar (mm3; WT=113±9, EC-SOD=118±11, p=0.89). Contralateral hemispheric CBF's were similar (ml/100g/min; WT=121±51, EC-SOD=125±44, p=0.89) Conclusions: No difference in the volume of brain at risk for infarct was found between transgenic EC-SOD and WT mice. It is likely that the mechanism of neuroprotection in EC-SOD mice is not related to the modulation of NO and its action on cerebrovascular smooth muscle, but more directly to EC-SOD scavenging of the damaging free radical SO.
Bart, R; Sheng, H; Oury, T; Crapo, J; Pearlstein, R; Warner, D
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